ELISA Handbook

G r a s p t h e P r o t e o m e ™

ELISAAND ELISPOT PRODUCTS

800-874-3723 • 815-968-0747 • www.piercenet.com

Table of ContentsELISA the Pierce Way ...........................................................2Introduction to ELISA ............................................................4

Typical ELISA Protocol ........................................................................4Types of ELISAs...................................................................................4Direct vs. Indirect Detection Techniques for ELISA .............................5Developing an ELISA ...........................................................................6

Selecting an ELISA Plate .......................................................8

Coated Microplates ............................................................10

Blocking and Washing .........................................................24

Primary and Secondary Antibodies..........................................32Affinity-Purified Primary Antibodies ..................................................32Antibody Conjugates and Enzyme Detection .....................................33Affinity-Purified Secondary Antibodies..............................................35

Antibody Labeling ..............................................................40Fluorescent Labeling..........................................................................41Enzyme Labeling................................................................................42

Choosing a Substrate ..........................................................46Horseradish Peroxidase Substrates...................................................47Alkaline Phosphatase Substrates.......................................................54

ELIFA System ...................................................................56

Bulk and Custom Offerings....................................................58

Recommended Reading.......................................................60

Enzyme-linked immunosorbent assays (ELISAs) are designed for detectingand quantitating substances such as peptides, proteins, antibodies and hormones. Othernames, such as enzyme immunoassay (EIA), are also used to describe the same process.In an ELISA, an antigen must be immobilized to a solid surface. The antigen is thencomplexed with an antibody that is linked to an enzyme. Detection is accomplished byincubating this enzyme-complex with a substrate that produces a detectable product. Themost crucial element of the detection strategy is a highly specific antibody-antigen interaction.

Most commonly, ELISAs are performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodiesand proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform, as firstdescribed by Eva Engvall, et al.1 Having the reactants of the ELISA immobilized to the microplate surface makes iteasy to separate bound from unbound material during the assay. This ability to wash away nonspecifically boundmaterials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation.

A detection enzyme may be linked directly to the primary antibody or introduced through a secondary antibody thatrecognizes the primary antibody. It may also be linked to a protein such as streptavidin if the primary antibody isbiotin-labeled. However the enzyme is incorporated, the most commonly used enzymes are horseradish peroxidase(HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespreadacceptance because of limited substrate options. These include β-galactosidase, acetylcholinesterase and catalase.A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The choice ofsubstrate depends upon the necessary sensitivity level of the detection and the instrumentation available for detection(e.g., spectrophotometer, fluorometer or luminometer).

Enzyme-linked immunospot assays (ELISPOTs) are a subclass of ELISAs that are used to count the number ofcytokine-producing cells within a cell suspension. The difference between the two is that in ELISA, the substancecontaining the “unknown” is stuck at the bottom of the well, whereas in ELISPOT the substance within the “unknown”is placed in the well after the bottom of the well has been coated with cytokine-specific antibody. In both cases, thewells are typically contained within a generic microplate.

ELISAAND ELISPOT PRODUCTS 1

2 800-874-3723 • 815-968-0747 • www.piercenet.com

ELISATHE PIERCE WAY

• StartingBlock™ Blocking Buffer in PBS (Product # 37538)and in TBS (Product # 37542)

• StartingBlock™ T20 Blocking Buffer (Contains 0.05% Tween®-20)in PBS (Product # 37539) or TBS (Product # 37543)

• SuperBlock® Buffer in PBS (Product # 37515) and in TBS(Product # 37535)

• SuperBlock® T20 Blocking Buffer (Contains 0.05% Tween®-20)in PBS (Product # 37516) or TBS (Product # 37536)

Block unoccupied sites.

• Casein in PBS (Product # 37528) andin TBS (Product # 37532)

• BSA in PBS (Product # 37525) and in TBS (Product # 37520)

• SEA BLOCK Buffer (Product # 37527)

• BLOTTO in TBS (Product # 37530)

• Metal Chelate Buffer (Product # 37547)

STEP 2. Blocking

STEP 3. Add sample and incubate

• 96-well Plates (Product #s 15041 and 15042)

• 8-well Strip Plates (Product # 15031)

• Strip Well Ejector (Product # 15033)

• Protein A (Product #s 15130 and 15132)

• Protein G (Product #s 15131 and 15133)

• Protein A/G (Product # 15138)

• Protein L (Product # 15190)

• Secondary Antibodies (Product #s 15134 and 15135)

• Anti-GFP (Product #s 15182, 15186 and 15188)

• NeutrAvidin™ Biotin-Binding Protein (Many options, see page 15-16)

• Streptavidin (Many options, see page 17-18)

Choose a plate.

• Sealing Tape for Plates (Product # 15036)

• Reagent Reservoirs (Product # 15075)

• DNA Coating Solution (Product # 17250)

Use a pre-coated plate for efficiency and consistency.

• Biotin (Product # 15151)

• Nickel Chelate (Product #s 15142, 15242 and 15342)

• Copper Chelate (Product #s 15143, 15146, 15147 and 15148)

• Glutathione (Product #s 15140, 15240 and 15340)

• Poly-D-Lysine (Product #s 15600 and 15608)

• Collagen I (Product #s 15610 and 15618)

• Maleic Anhydride (Product #s 15110, 15112, 15100 and 15102)

• Maleimide (Product # 15150)

Pre-coated plates

STEP 1. Selecting an ELISA plate

3

• Phosphate Buffered Saline (PBS, Product # 28372)

• Tris Buffered Saline (TBS, Product #s 28376 and 28379)

• Modified Dulbecco’s PBS (Product # 28374)

• Carbonate-Bicarbonate Buffer Packs (Product # 28382)

[Skip this step if you use StartingBlock™ T20 Blocking Buffer in PBS(Product # 37539) or TBS (Product # 37543) or SuperBlock® T20 BlockingBuffer in PBS (Product # 37516) or TBS (Product # 37536). These buffersalready contain Tween®-20 Detergent at optimized concentrations.]

Surfact-Amps® Brand Detergents containing:

• Tween®-20 (Product # 28320) and Tween®-80 (Product # 28328)

Choose a buffer.

• MES Buffered Saline (Product # 28390)

• BupH™ Borate Buffer Packs (Product # 28384)

• BupH™ Citrate-Carbonate Buffer Pack (Product # 28388)

Reduce nonspecific binding.

• Triton® X-100 (Product # 28314) and Triton® X-114 (Product # 28332)

• Nonidet P-40 (Product # 28324)

• Brij®-35 (Product # 28316) and Brij®-58 (Product # 28336)

Add detergent to buffers

STEP 4. Formulate wash buffers

For a complete list, visit the antibody selection guide on our website (www.piercenet.com) accessible under the Products tab.

For direct detection methods we offer:

• Monoclonal Antibodies

• Fluorescent Probes and Labeling Kits

• Enzyme Labeling Kits

For indirect detection methods we offer:

• Biotinylation Kits

Incubate the membrane with antibodies.

• Protein A, Protein G and Protein L labeled with fluorescein, rhodamine, HRP, AP or biotin

• Avidin, Streptavidin and NeutrAvidin™

Biotin-Binding Protein labeled with fluorescein, rhodamine, HRP or AP

• Secondary antibodies labeled with fluorescein, rhodamine, HRP, AP or biotin

STEP 5. Primary and Secondary Antibodies

HRP

Chemiluminescent Substrates:

• SuperSignal® ELISA Pico Chemiluminescent Substrate (Product # 37070)

• SuperSignal® ELISA Femto Chemiluminescent Substrate (Product # 37075)

Chemifluorescent Substrate:

• QuantaBlu™ Fluorogenic Peroxidase Substrate (Product #s 15162 and 15169)

Add the detection reagent.

Colorimetric Substrates:

• TMB Substrates (Product #s 34021, 34022, 34024 and 34028)

• ABTS Substrates (Product #s 34026 and 37615)

• OPD Substrate (Product #s 34005 and 34006)

• PNPP Substrates (Product #s 34045, 34047, 37620 and 37621)

STEP 6. Enzyme Substrates for Detection

HRP

SuperSignal®

Substrate

Typical ELISA Protocol

Coating antibody or antigen onto the microplate1. Dilute the protein to be coated to a concentration of

2-10 µg/ml in a buffer such as PBS or Carbonate-Bicarbonate and add 100 µl of this solution per well.

2. Incubate for 18-20 hours at room temperature or 4°C.3. Block unoccupied sites with a blocking agent

(200-300 µl/well) such as StartingBlock™ Blocking Buffer.4. Store plate at 4°C with a dessicant for future use.

Perform assay5. Add sample to be tested (50-100 µl/well) and incubate

for 1 or more hours.6. Wash using PBS with 0.05% Tween®-20 or TBS with

0.05% Tween®-20.7. Add enzyme-antibody conjugate (100-200 µl/well) diluted

in blocking buffer and incubate for 1 hour.8. Wash again.9. Add substrate.

10. Detect.

Types of ELISAs

The most commonly used ELISA assay format is the sandwichassay (Figure 1). This type of assay is called a “sandwich” assaybecause the analyte to be measured is bound between twoantibodies – the capture antibody and the detection antibody.The sandwich format is used because it is sensitive and robust.2Competitive assays are often used when the antigen is smalland has only one epitope, or antibody-binding site.3 Often theantigen is labeled instead of the antibody. Unlabeled antigenand the labeled antigen compete for binding to the captureantibody and a decrease in signal indicates the presence ofthe antigen in the sample. ELISPOT assays are a form ofsandwich ELISAs.

Either monoclonal or polyclonal antibodies may be used as thecapture and detection antibodies in sandwich ELISA systems.Monoclonals have an inherent monospecificity toward a singleepitope that allows fine detection and quantitation of smalldifferences in antigen. A polyclonal is often used as the captureantibody to pull down as much of the antigen as possible. Thena monoclonal is used as the detecting antibody in the sandwichassay to provide improved specificity.

An important consideration in designing a sandwich ELISA isthat the capture and detection antibodies must recognize twonon-overlapping epitopes. When the antigen binds to the captureantibody, the epitope recognized by the detection antibody mustnot be obscured or altered. Capture and detection antibodies thatdo not interfere with one another and can bind simultaneouslyare considered a matched pair and are suitable for developinga sandwich ELISA.

Another design consideration in choosing antibodies is cost.A polyclonal antibody is generally less expensive (~five-fold)to produce than a monoclonal. The specificity gained by usingmonoclonals for both the capture and detecting antibody mustbe weighed against the cost and time required for producingtwo monoclonal antibodies. Preparing a “self-sandwich” ELISAassay, in which the same antibody is used for the capture anddetection, can limit the dynamic range and sensitivity of thefinal ELISA.

4

INTRODUCTIONTO ELISA


Direct Assay

E

Ag

Primaryantibodyconjugate

Biotinylatedprimaryantibody

Indirect Assay

Primaryantibody

Secondaryantibodyconjugate

Avidin-Biotin Complex Capture Assay “Sandwich” or ELISPOT

Primaryantibody #1

Primaryantibody #2

Substrate

Substrate

Substrate

Substrate

Substrate

SubstrateSubstrate

E

E

E

E

Ag

Ag

Ag

E

E

E

E

Figure 1. Immunoassay formats.

5INTRODUCTIONTO ELISA

Direct vs. Indirect Detection Techniques for ELISA

The direct detection method originated in the 1940s whenCoons and colleagues labeled antibodies with a fluorescent tagto mark tissue antigens.4 In this technique, a labeled primaryantibody reacts directly with the antigen (Figure 1). Directdetection is not widely used in ELISAs, but is quite commonfor immunohistochemical staining of tissues and cells.

Advantages of direct detection• Quick methodology, because only one antibody is used.• Cross-reactivity of secondary antibody is eliminated.

Disadvantages of direct detection• Immunoreactivity of the primary antibody may be reduced

as a result of labeling.• Labeling of every primary antibody is time-consuming

and expensive.• No flexibility in choice of primary antibody label from

one experiment to another.• Little signal amplification.

The indirect, two-step method uses a labeled secondary antibodyfor detection (Figure 1). This was first described by Weller andCoons in 1954 and is still a popular method.5 First, a primaryantibody is incubated with the antigen. This is followed by

incubation with a labeled secondary antibody that recognizesthe primary antibody. For ELISA it is important that the antibodyenzyme conjugate is of high specific activity. This is achievedwhen the antibody is affinity-purified and the enzymeconjugation chemistry preserves antibody specificity as wellas enzyme activity. All Pierce antibody-enzyme conjugatesfulfill these requirements.

Advantages of indirect detection• A wide variety of labeled secondary antibodies are

commercially available.• Versatility, because many primary antibodies can be made in

one species and the same labeled secondary antibody can beused for detection.

• Immunoreactivity of the primary antibody is not affected by labeling.

• Sensitivity is increased because each primary antibodycontains several epitopes that can be bound by the labeledsecondary antibody, allowing for signal amplification.

• Different visualization markers can be used with the sameprimary antibody.

Disadvantages of indirect detection• Cross-reactivity may occur with the secondary antibody,

resulting in nonspecific signal.• An extra incubation step is required in the procedure.

Developing an ELISA

Optimizing immunoreagent concentrations and dilutionsThe goals in developing an ELISA assay are 1) to achieve thebest signal:noise ratio for the sensitivity level desired, 2) to havea robust, reproducible assay for the sample being tested and3) to be able to measure the antigen over a biologically relevantassay range (dynamic range). Therefore, ideal concentrationsof each assay reagent must be established empirically. Thesignal generated by a sample containing analyte, relative to thesignal of the same sample without analyte, is the signal:noiseratio. As the signal:noise ratio increases, the assay becomesbetter at measuring small amounts of antigen.

Dilution ranges for assay reagents can vary widely, dependingupon the detection system used. For example, most ELISAprotocols based on enzyme-antibody conjugates using a colori-metric substrate recommend a 1:5,000 dilution (from a 1 mg/mlstock concentration) of the conjugate. But this ELISA maywork equally well at 1:2,000 or 1:20,000 dilution. To establishthe optimal dilutions, a checkerboard titration, also called atwo-dimensional serial dilution, is performed. A checkerboardtitration is a single experiment in which the concentration oftwo components is varied in a way that will result in a pattern(Figure 2). This method is used to optimize reagent concentra-tions when performing an indirect ELISA that uses a labeledsecondary antibody. For example, the primary antibody is seriallydiluted across the plate, and the enzyme-labeled secondaryantibody is serially diluted down the plate. This design permitsanalysis of different concentrations of the two reagents in eachwell to obtain the best signal:noise ratio.

Figure 2. A checkerboard titration using a precipitating substrate.

A more complex checkerboard titration for developing an ELISAcan be seen in Figure 3. Here the coating or capture antibodyis titered first by using high concentrations of biotinylateddetecting antibody and streptavidin-HRP conjugate. Once thesignal falls within the desired detection range (dynamic range),the checkerboard matrix in Figure 3 can be tested to optimizethe concentrations of the biotinylated detecting antibody andstreptavidin-conjugate.

1:1 1:2 1:4 1:8 1:16 1:32 1:64

1:1

1:2

1:4

1:8

1:16

1:32

1:64

1 2 2 3 4 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

Primary Antibody Dilution

Labe

led

Seco

ndar

y An

tibod

y Di

lutio

n

6

INTRODUCTIONTO ELISA


3B. When dividing the mean O.D. for the 80 pg/ml standard by the O.D. forthe zero standard, 250 ng/ml produced the greatest signal:noise ratio,suggesting the antibody is on a plateau. Using this detecting antibody at250 ng/ml would provide optimal sensitivity, assay reproducibility andsample recovery.

3C. The standard curve is within the desired O.D. range at the 1:32,000dilution of HRP-conjugated streptavidin. For increased sensitivity with asmaller assay range, a higher concentration of HRP-conjugated streptavidinmay be selected. Poly-HRP streptavidin may also be used.

HRP-Conjugated Streptavidin Titration Curves(Biotin-Labeled Antibody at 60 ng/ml)Ab

sorb

ance

at 4

50 n

m-5

50 n

m

HumanTNFα (pg/ml)

1:32,0001:16,0001:8,000

0 500 1,000 1,500 2,000

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

Human TNFα Detecting Antibody Titration Curves(HRP-Conjugated Streptavidin at 1:32,000)

Abso

rban

ce a

t 450

nm

-550

nm

HumanTNFα (pg/ml)

250 ng/ml500 ng/ml

125 ng/ml60 ng/ml

0 500 1,000 1,500 2,000

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

Figure 3. Checkerboard matrix: example of human TNFα detecting antibody and enzyme titration.

3A. The plate is divided into four equal quarters for four different detecting antibody concentrations. For each detecting antibody concentration, three differentenzyme concentrations are tested on three different levels of standards, plus a zero. Prior to this checkerboard titration, a coating titration was performed to deter-mine the optimal concentration for the coating antibody.

From this checkerboard matrix, the following curves are generated:

0 pg/ml 0.032 0.031 0.028 0.030 0.029 0.028 0.047 0.047 0.042 0.039 0.038 0.037 80 pg/ml 0.225 0.219 0.208 0.207 0.167 0.175 0.402 0.402 0.363 0.369 0.287 0.294 400 pg/ml 0.555 0.545 0.605 0.561 0.438 0.431 1.603 1.108 1.034 1.032 0.742 0.768 2,000 pg/ml 2.870 2.620 2.494 2.476 2.037 1.770 **** **** **** **** 2.709 2.866 0 pg/ml 0.041 0.040 0.032 0.031 0.031 0.030 0.074 0.067 0.060 0.059 0.052 0.048 80 pg/ml 0.337 0.335 0.293 0.296 0.229 0.232 0.448 0.411 0.436 0.419 0.357 0.347 400 pg/ml 0.831 0.886 0.838 0.829 0.604 0.613 1.466 1.348 1.243 1.255 0.891 0.884 2,000 pg/ml 3.720 **** 3.263 3.454 2.373 2.468 **** **** **** **** 3.187 3.141

(Coating Antibody at 2 µg/ml)

HRP-Conjugated Streptavidin Dilution

Stan

dard

s

Biotin-LabeledDetecting AntibodyConcentration

60 ng/ml

125 ng/ml

250 ng/ml

500 ng/ml

1:8,000 1:16,000 1:32,000 1:8,000 1:16,000 1:32,000

7INTRODUCTIONTO ELISA

References1. Engvall, E. and Perlmann, P.O. (1971). Immunochemistry 8, 871-875.2. Palomäki, P. (1991). J. Immunol. Method 145, 55-63.3. Rao, P.N. and Taraporewala, I.B. (1992). Steroids 57, 154-161.4. Coons, A.A., et al. (1942). J. Immunol. 45, 159-170.5. Weller, T.H. and Coons, A.H. (1954). Proc. Soc. Exp. Biol. (New York) 86, 789-794.

When binding an anti-body, select a microplatewith high-bindingcapacity. Choose amicroplate with a

minimum protein-binding capacity of 400 ng/cm2. It is alsoimportant that the CV value (coefficient of variation) of theprotein binding be below 5%. Pierce ELISA plates undergoextensive quality testing and controlled manufacture and areguaranteed to have CVs less than 5%. Use Reacti-Bind™ 8-WellStrip Plates (Product # 15031) when partial-plate assays areperformed frequently. The choice of plate color depends upon

the signal being detected. Clear polystyrene plates are usedfor colorimetric signals and black or white opaque plates areused for fluorescent and chemiluminescent signals.

Characteristics:Antibody Binding: High, 400 ng IgG/cm2 (1 cm2 = 100 µl vol.)Binding Interaction: Hydrophobic/Ionic (-)Performance Certified: Yes. Well-to-well, CV ≤ 3%Well Shape (bottom): FlatMaximum Well Volume: 360 µlPackaging: 1 plate per sealed trayUnits per package: 100 plates

ImmunoWare™ Reagent ReservoirsAllows easy dispensing of reagents by multi-channel pipettors.

Highlights:• Sterile, sturdy reservoirs

are molded from high-impact polystyrene

• Reservoirs facilitaterepeated pick up ofreagents by multi-channel pipettors fordelivery to 96-well plates

Ordering InformationU.S.

Product # Description Pkg. Size Price15075 ImmunoWare™ Reagent Reservoirs 200/pkg. $ 99

Sterile, 50 ml capacity

Sealing Tape for 96-Well Plates

Highlight:• Seal plates quickly and easily to allow mixing or prevent

evaporation


Product # Description Pkg. Size Price15036 Sealing Tape for 96-Well Plates 100/pkg. $ 48

Pre-cut pressure-sensitive sealing tape

Reacti-Bind™ 96-Well PlatesEnhance antibody-binding properties in your ELISA applications.

Highlights:• Plates are made of

specially formulatedpolystyrene and treatedto produce high antibody-binding characteristics

• Each lot is certified toensure adherence tostrict performance criteria

ReferenceFoster, B.A., et al. (1999). Science 286, 2507-2510.

Characteristics:Antibody Binding: High, 400 ng IgG/cm2 (1 cm2 = 100 µl vol.)Binding Interaction: Hydrophobic/IonicPerformance Certification: Well-to-well, CV ≈ 3%Well Shape (bottom): FlatMaximum Well Volume: 360 µlPackaging: 1 plate per sealed tray


Product # Description Pkg. Size Price15041 Reacti-Bind™ 96-Well Plates – Corner Notch 100/pkg. $27815042 Reacti-Bind™ White Opaque 96-Well Plates 25/pkg. $ 84

8

SELECTINGAN ELISA PLATE

Reacti-Bind™ 8-Well Strip PlatesNo more loose-fitting strips!

Highlights:• Special optically clear

polystyrene formulation• High antibody-binding

surface improves theoverall performance in immunoassay applications

• Plates are certified for performance

• Consistent lower coefficients of variation• Eight-well strips fit snugly in the plate frame, virtually elimi-

nating strip loss while in use – even if turned upside down• Strip guides help to avoid misorientation of the strip when

returned to the holder• Strips are gently removed from the frame with a strip well ejector• Wells can be easily separated from the strips with a gentle

twist, allowing you to work with individual wells or strips ofless than eight wells

Characteristics:Antibody Binding: High, 400 ng IgG/cm2 (1 cm2 = 100 µl vol.)Binding Interaction: Hydrophobic/IonicPerformance Certification: Well-to-well, CV ≈ 3%Well Shape (bottom): FlatMaximum Well Volume: 360 µlPackaging: 5 plates per sealed tray


Product # Description Pkg. Size Price15031 Reacti-Bind™ 8-Well Strip Plates – 100/pkg. $462

Corner NotchIncludes one Strip Well Ejector per package.

Accessory

15033 Reacti-Bind™ Strip Well Ejector 1/pkg. $ 29

Reacti-Bind™ DNA Coating SolutionQuickly deposit DNA onto a plastic surface for hybridization.

Reacti-Bind™ DNA Coating Solution is the latest answer forefficient DNA surface coating of microplates and micro-sample tubes.

Highlights:• No more overnight incubations or heating microplates

and waiting• DNA Coating Solution promotes efficient immobilization

of DNA onto microplates – hybridization efficiency is relatedto efficiency of DNA surface immobilization.1 This solutionpromotes high-efficiency coating of DNA onto polystryene or polypropylene surfaces

• Target DNA coated onto microplates permits speed andflexibility in probing

• DNA coated onto plastic surfaces is stabilized sufficiently to allow storage for months prior to use

• DNA Coating Solution autoclaved to inactivate DNase

References1. Hirayama, H., et al. (1996). Nucleic Acids Res. 24, 4098-4099.van Gijssel, H.E., et al. (2002). Cancer Epidem. Biomarker Prev. 11, 1622-1629.


Product # Description Pkg. Size Price17250 Reacti-Bind™ DNA Coating Solution 100 ml $ 63

Sufficient for coating 5 x 96-well microplates (at 200 µl/well).

9SELECTINGAN ELISA PLATE

There are several important points to consider when coatingan ELISA plate. It is important to ensure that the coating solu-tion is free of detergents, because detergents often compete forbinding and cause low and/or uneven binding. For competitiveassays, a lower coating concentration usually is chosen toensure that the antibody is the limiting factor. Oddly, excessiveconcentrations of coating protein occasionally lead to lessbinding, a phenomenon known as the “hook” effect. A typicalprotein-coating solution concentration of 2-10 µg/ml can beused as a starting point for successful plate-coating.

Smaller molecules such as peptides and many drugs requirechemically activated microplates such as Reacti-Bind™ MaleicAnhydride Activated Plates (Product # 15100 or 15110) or

Maleimide Activated Plates (Product # 15150) to achieve covalentattachment to the plate surface. The maleic anhydride groupreacts with primary amines (lysine, N-terminus) and themaleimide reacts with sulfhydryl groups (–SH, reduced cysteineresidues), forming a covalent bond (Figure 4). To increase thedynamic range of an assay for small molecules, they may bebiotinylated and attached to a Reacti-Bind™ NeutrAvidin™ HBC(High Binding Capacity) Plate or a Reacti-Bind™ StreptavidinHBC Plate (Figure 5). These plates allow binding of morebiotinylated oligos, peptides, etc. to the surface of the well,and are provided as clear, black or white opaque plates andin 96- or 384-well plate formats.

H2N —

pH ≥ 7.2

SH —

pH > 6.5-7.5

Reaction Scheme for Coupling both Large and Small Amine-Containing Molecules

= Protein, peptide or other ligand

Maleic Anhydride shown anchored to polystyrene well of a microplate.Both the well wall and base are activated.

Stable amide bond formation withamines, with a resulting hydrophilic well surface.

Reaction Scheme for Coupling both Large and Small Sulfhydryl-Containing Molecules

Maleimide shown anchored to polystyrene well of a microplate.Both the well wall and base are activated.

Stable thioether bond formation with sulfhydryls, with a resulting hydrophilic well surface.


R

R

R

R

R

R

O

O

C

C

N

C C

O( )n

N

SO

O

( )n

O

OOCC

( )n

CC

N

O

O

( )n

Figure 4. Coupling reactions of Reacti-Bind™ Activated Plates.

10

COATEDMICROPLATES


Figure 5. Comparison of NeutrAvidin™ High Binding Capacity (HBC)Coated Plate, NeutrAvidin™ Regular Binding Capacity (RBC) CoatedPlates and a competitor’s Streptavidin Coated High Binding CapacityPlates (CHC). Plates were incubated with various dilutions of biotinylated,phosphorylated peptide. After washing, the plates were incubated withmouse anti-phosphotyrosine antibody (1:1,000) and then detected usingan anti-mouse-FITC conjugate (1:667). The Y-axis is described as thesignal:noise (S/N) ratio.

The initial step in many microplate-based assays involves passiveadsorption of proteins to the plate wells. Several problemsarise from passive adsorption, including improper orientation,denaturation, poor immobilization efficiency and binding ofcontaminants along with the target molecule. Reacti-Bind™

Coated Plates from Pierce are designed to circumvent theseproblems. Antibodies can be covalently attached to a microplatethrough the Fc region using Protein A, G, or A/G coated plates,which orients them properly and preserves their antigen-binding

capability. Fusion proteins can be attached to a microplate inthe proper orientation using Glutathione, Metal Chelate orAnti-GFP Coated Plates. Because the polystyrene surface ofReacti-Bind™ Coated Plates is precoated, immobilized proteinsare physically separated from the surface and protected fromits denaturing effects. Peptides and other small molecules,which are typically difficult to immobilize, can be biotinylatedand attached to a streptavidin or NeutrAvidin™ Coated Platewith high efficiency. Precoated plates bind selectively to thedesired target proteins, minimizing any contamination fromother molecules that are present in the preparation.

Studies have shown that the binding of antibodies to a microplatesurface causes some denaturation.1 This can be solved inseveral ways. Antibodies can be bound to a binding protein,such as Protein A, Protein G, Protein A/G or Protein L, that iscoated on the plate. Alternatively, the antibody may be biotiny-lated and immobilized onto streptavidin or NeutrAvidin™ CoatedPlates. Either method physically separates the antibody fromthe surface of the plate and can result in proper orientation ofthe antigen-binding sites for better assay performance.

Histidine-, green fluorescent protein (GFP)- and glutathioneS-transferase (GST)-tagged fusion proteins are popular formanipulating recombinantly expressed proteins and developingELISAs to detect the expression levels of these tagged proteinsis simplified with microplates that are precoated with the ligandfor capturing the fusion protein to be measured. Reacti-Bind™

Precoated Plates save time in developing ELISAs by 1) capturingthe fusion protein from a crude cell lysate, 2) separating thefusion protein from the surface of the microplate to preventdenaturation and 3) properly orienting the fusion protein fordetection. Table 1 lists the variety of Reacti-Bind™ Coated Platesavailable and the ligands they bind.

HBC

RBC

CHC

Biotinylated Phosphopeptide (pM/well)

S/N

Ratio

10

8

6

4

2

00 5 10 15

Phosphopeptide Detection Assay Comparisonof Biotin-Binding Protein Coated Plates

11COATEDMICROPLATES

12

COATEDMICROPLATES

Table 1. Reacti-Bind™ Coated Polystyrene Microplates

Protein A, G or A/G For binding antibodies via their Fc regionsProtein L For binding Fab antibody fragments and single-chain variable fragments (ScFvs) through the kappa light chainSecondary Antibodies For binding antibodies, as an alternative to Protein A, G or LNeutrAvidin™ Protein or Streptavidin For binding biotinylated proteins, peptides or nucleic acids; also available in black or white opaque microplatesBiotin For binding avidin, streptavidin or NeutrAvidin™ Biotin-Binding ProteinHisGrab™ (Ni2+) or Glutathione For binding recombinantly expressed proteins containing polyhistidine or glutathione S-transferaseMaleic Anhydride For binding both large and small amine-containing moleculesMaleimide Activated For binding sulfhydryl-containing moleculesAnti-GFP For capturing proteins expressing green fluorescent protein (GFP) tagAnti-GST For capturing proteins expressing glutathione S-transferase (GST)

Figure 6. Poly-D-Lysine Coated 96-Well Plates comparison (Pierce vs.Competitor B). Poly-D-Lysine plates (96-well white with clear bottom) fromPierce and Competitor B were tested for their ability to support attachmentand spreading of rat cerebellar granule (RCG) cells. The graph representscell proliferation assay results following a 24-hour cell incubation.

Cell-based assays have become popular for detecting the effectsof various drugs, toxins or growth factors on cellular growth.To enhance cell attachment to the surface of a 96-well plate,CellScreen™ Plates are coated with collagen or poly-D-lysine(Figure 6) and are irradiated to prevent bacterial growth.CellScreen™ Plates are offered in a variety of formats (96- or384-well) for flexible assay design.

Pierce also offers a custom plate-coating service for largebatches of coated plates. Our automated plate-coating technologyproduces consistently coated plates that will meet your qualityspecifications. Take advantage of our coating expertise toexpedite the development of your plate-based assay. Seepages 58-59 for more information on custom-coated plates.

Reference1. Schwab, C. and Bosshard, H.R. (1992). J. Immunol. Method 147, 125-134.

Pierce

Competitor B

Abso

rban

ce a

t 490

nm

Cells/Well

0.00 40,00010,000 20,000 30,000

0.4

0.2

0.6

0.8


Reacti-Bind™ Protein A, G and A/G Coated PlatesBind antibodies through their Fc portion, correctly orienting them for maximum antigen capture.

Highlights of using Reacti-Bind™ Plates forantibody capture:• Retain antibody activity,

which can be lost whenantibodies are immobilizedby passive adsorption

• Orient antibodies formaximum antigen-bindingcapacity

• Immobilize antibodies without prior purification• Ensure minimal variation (< 5% well-to-well) from

consistent coating • Reduce nonspecific binding because plates are pre-blocked

with SuperBlock® Blocking Buffer• Perform multiple immunoprecipitations • Binds up to 2.5 µg antibody/well (100 µl coating volume)

Protein A/G• Consists of a fusion protein containing four antibody-binding

sites from Protein A and two from Protein G • Binds to the broadest spectrum of antibodies because it

combines the specificities of Protein A and Protein G• Binds to Fc region of antibodies, ensuring optimal orientation• Binds well using a wide range of pH

Protein A• Binds strongly to IgG from rabbit, guinea pig, pig, dog and

rhesus monkey• Binds strongly to mouse IgG2a, IgG2b and IgG3

• Binds to Fc region of antibodies for optimal orientation

Protein G• Binds strongly to IgG from many species including human,

mouse, rabbit, sheep and goat• Binds only to IgG – no cross-reactivity with other

antibody classes• Binds to Fc region of antibodies for optimal orientation

Figure 7. Properly oriented antibodies retain higher activity.

ReferenceDesai, S. and Hermanson, G. (1997). Previews 1(3), 2-7.Protein A Coated Plate ReferencesAsthagiri, A.R., et al. (1999). J. Biol. Chem. 274, 27119-27127.Lawrenson, I.D., et al. (2002). J. Cell Sci. 115, 1059-1072. Protein G Coated Plate ReferencesLai, Z., et al. (2002). Proc. Natl. Acad. Sci. 99, 14734-14739. Rauch, J., et al. (2003). J. Biol. Chem. 278, 47508-47515.


Product # Description Pkg. Size Price15138 Reacti-Bind™ Protein A/G Coated 5 plates $170

8-Well Strip Plates15130 Reacti-Bind™ Protein A Coated 5 plates $141

96-Well Plates15132 Reacti-Bind™ Protein A Coated 5 plates $163

8-Well Strip Plates15131 Reacti-Bind™ Protein G Coated 5 plates $156

96-Well Plates15133 Reacti-Bind™ Protein G Coated 5 plates $168

8-Well Strip Plates

Passively Bound Antibody

Protein G

Abso

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t 450

nm

Antibody Added per Well (ng)0 0.16 0.312 0.625 1.25 2.5 5 10

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

13COATEDMICROPLATES

14

COATEDMICROPLATES

Reacti-Bind™ Secondary Antibody Coated PlatesFor species-specific antibody capture.

Highlights:• Prevents antibody denaturation as a result of direct binding

to polystyrene• Unlike Protein A or Protein G Plates, these plates bind only

to target species• Antibody-binding capacity is higher than direct adsorption

onto polystyrene• Pre-blocked with SuperBlock® Buffer to reduce

nonspecific binding

ReferencesGartner, W., et al. (2001). Cereb Cortex. 11, 1161-1169. Wagner, L., et al. (2000). J. Biol. Chem. 275, 24740-24751.


Product # Description Pkg. Size Price15134 Reacti-Bind™ Goat Anti-Mouse Coated 5 plates $130

Clear 96-Well Plates15135 Reacti-Bind™ Goat Anti-Rabbit Coated 5 plates $130

Clear 96-Well Plates

Reacti-Bind™ Protein L Coated PlatesGreat for binding ScFv and Fab fragments.

Protein L is an immunoglobulin-binding protein that has theunique ability to bind through kappa light chain interactionswithout interfering with an antibody’s antigen-binding site.This gives Protein L the ability to bind a wider range of Igclasses and subclasses than any other antibody-binding proteinssuch as Protein A or Protein G. This also gives Protein L theunique ability to bind single-chain variable fragments (ScFvs)and Fab fragments.

Highlights:• Binds to all classes of Ig (IgG, IgM, IgA, IgE and IgD)• Binds to the VL region of kappa light chains (human I, III and IV

and mouse I) without interfering with antigen-binding sites• Binds ScFvs• Does not bind Bovine, Goat or Sheep Igs• Binds weakly to Rabbit Igs• Pre-blocked with SuperBlock® Buffer to reduce

nonspecific binding

ReferencesÅkerström, B. and Björck, L. (1989). J. Biol. Chem. 264, 19740-19746.Björck, L. (1988). J. Immunol. 140, 1194-1197.Kastern, W., et al. (1992). J. Biol. Chem. 267, 12820-12825.Nilson, B.H.K., et al. (1993). J. Immunol. Method 164, 33-40.Rhee, J., et al. (2001). J. Biol. Chem. 276, 6640-6644.


Product # Description Pkg. Size Price15190 Reacti-Bind™ Protein L Coated 5 plates $168

96-Well Plates


Reacti-Bind™ NeutrAvidin™ Coated PlatesThe high affinity of avidin for biotin, without the nonspecific binding problems.

Highlights: • Easy and gentle immobilization of biotin-containing conjugates• Lowest nonspecific binding properties of all biotin-binding

proteins• NeutrAvidin™ Biotin-Binding Protein has no carbohydrate and

an isoelectric point of 6.3• No denaturing of the protein component of a conjugate upon

binding to the plate• Ideal for binding small hydrophilic molecules (e.g., peptides)

that typically exhibit poor binding directly to polystyrene• Pre-blocked with your choice of Blocker™ BSA or SuperBlock®

Blocking Buffer• Available in 96-well and 384-well formats

Isoelectric Contains NonspecificProtein Point Carbohydrate BindingAvidin 10-10.5 Yes HighStreptavidin 5 No LowNeutrAvidin™ Protein 6.3 No Ultralow

Figure 8. Purified p60c-sr activity detection with TK peptide 2. Biotinylatedtyrosine kinase peptide 2 was added to Reacti-Bind™ NeutrAvidin™ CoatedPlates and incubated for 30 minutes. Wells were washed; samples containingp60c-src tyrosine kinase were added to phosphorylate the tyrosine residueon the peptide. Anti-phosphotyrosine monoclonal antibody conjugated toHRP was added. Tyrosine kinase activity was detected by 1-Step™ TurboTMB Substrate. Kinase activity was quantitated by comparison with astandard curve generated using the phosphorylated form of the samepeptide substrate.

NeutrAvidin™ Coated Plate Characteristics96-Well Plate 384-Well Plate

Binding Capacity 15 pmoles/well 10 pmoles/wellCoat Volume 100 µl/well 50 µl/wellBlocking Volume 200 µl/well 100 µl/well

ReferencesBrett, P.J., et al. (2002). J. Biol. Chem. 277, 20468-20476. Denlinger, L.C., et al. (2001). J. Immunol. 167, 1871-1876. Guixiang Dai, G., et al. (2002). J. Biol. Chem. 277, 161-168. Patil, S., et al. (1999). J. Biol. Chem. 274, 28575-28583. Singh, Y., et al. (1999). Infect. Immun. 67, 1853-1859.


Product # Description Pkg. Size Price15129 Clear 96-Well Plates with 5 plates $123

SuperBlock® Blocking Buffer15123 Clear 96-Well Plates with Blocker™ BSA 5 plates $12315128 Clear 8-Well Strip Plates with Blocker™ BSA 5 plates $17215127 Clear 8-Well Strip Plates with 5 plates $172

SuperBlock® Blocking Buffer15116 White 96-Well Plates with 5 plates $135

SuperBlock® Blocking Buffer15117 Black 96-Well Plates with 5 plates $135

SuperBlock® Blocking Buffer15400 Clear 384-Well Plates with 5 plates $262



SuperBlock® Blocking Buffer15115 Reacti-Bind™ Biotin Binding Plate 4 plates $112

Sample PackOne each of the following plates:Product #s 15120, 15121, 15127 and 15128

Abso

rban

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t 450

nm

Units src kinase0.00 0.05 0.10 0.15

1.5

1.0

0.5

0

15COATEDMICROPLATES

Reacti-Bind™ Anti-GFP Coated PlatesCreate more assay possibilities by capturing GFP fusion proteins on coated plates.

Highlights:• Unique anti-GFP antibody binds all variants of GFP fusion proteins • Coating is compatible with Poppers™ Cell Lysis Reagents and

B-PER®, M-PER® and Y-PER® Extraction Reagents• Directly detect GFP-fusion proteins on white plates without

interference from cellular autofluorescence or quenching agents• Pre-blocked to save assay development time• GFP excitation: 395 nm; emission 509 nm


Product # Description Pkg. Size Price15182 Black 96-Well Plates with Blocker™ BSA 5 plates $13515186 Clear 96-Well Plates with Blocker™ BSA 5 plates $13515188 Clear 8-Well Strip Plates with Blocker™ BSA 5 plates $141

16

COATEDMICROPLATES

Reacti-Bind™ NeutrAvidin™ High Binding Capacity (HBC) Coated PlatesUnique technology for improved assay precision.

Pierce’s new patent-pending plate-coating technology offers aNeutrAvidin™ HBC Plate with a wider detection limit than ourregular binding capacity plates. The standard curve exhibitsgreater linearity for detecting small biotinylated molecules suchas peptides (Figure 9) and oligonucleotides, resulting in greaterassay precision. Switch your assay over to Reacti-Bind™

NeutrAvidin™ HBC Coated Plates for binding small biotinylatedligands and see the difference for yourself.

Highlights: • Unique plate-coating technology – results in high loading of

NeutrAvidin™ Biotin-Binding Protein/well• Improved sensitivity – less nonspecific binding for improved

signal:noise ratios• Broader dynamic range – extends the quantitative range so

there is no need for dilutions• Save time – pre-blocked plates to reduce the number of

assay steps• Flexible assay formats – coated plates offered in 96- and

384-well formats and in different colors

Figure 9. Phosphopeptide detection assay comparison of biotin-bindingprotein-coated plates. Comparison of NeutrAvidin™ High Binding Capacity(HBC) Coated Plate, NeutrAvidin™ Regular Binding Capacity (RBC) CoatedPlates and a competitor’s Streptavidin Coated High Binding CapacityPlates (CHC). Plates were incubated with various dilutions of biotinylated,phosphorylated peptide. After washing, the plates were incubated withmouse anti-phosphotyrosine antibody (1:1,000) and then detected usingan anti-mouse-FITC conjugate (1:666). The Y-axis is described as thesignal:noise (S/N) ratio.

Reacti-Bind™ NeutrAvidin™ HBC Coated Plate Characteristics96-Well Plate 384-Well Plate



Product # Description Pkg. Size Price15507 Clear 96-Well Plates with SuperBlock® 5 plates $135

Blocking Buffer*15508 Clear 8-Well Strip Plates with SuperBlock® 5 plates $182

Blocking Buffer*15509 White 96-Well Plates with SuperBlock® 5 plates $146

Blocking Buffer*15510 Black 96-Well Plates with SuperBlock® 5 plates $146

Blocking Buffer*15511 Clear 384-Well Plates with SuperBlock® 5 plates $285



Blocking Buffer** U.S. patent pending on high-binding capacity plate-coating technology.

S/N

Ratio

Biotinylated Phosphopeptide (pM/well)

CHCRBCHBC

0 5 10 15

10

8

6

4

2

0


Reacti-Bind™ Biotin Coated PlatesA simple format for testing the efficiency of avidin, streptavidin or NeutrAvidin™ Protein conjugations.

Reacti-Bind™ Biotin Coated Plates can be used in any immunoassaywith NeutrAvidin™ Biotin-Binding Protein, streptavidin, avidinor other biotin-binding proteins.

Highlights:• Biotin group accessible for binding avidin, streptavidin or

NeutrAvidin™ Biotin-Binding Protein• Pre-blocked to reduce nonspecific binding • Strip well plate format for the ultimate in convenience• Coat volume: 200 µl• Blocking volume: 300 µl


Product # Description Pkg. Size Price15151 Reacti-Bind™ Biotin Coated Clear 8-Well 5 plates $123

Strip Plates

Reacti-Bind™ Streptavidin Coated PlatesThe specific binding affinity of streptavidin for biotin – in a microplate.

Highlights:• Easy and gentle immobilization of biotin-containing conjugates• Low nonspecific binding• No denaturing of the protein component of a conjugate

upon binding• Ideal for binding small biotinylated hydrophilic molecules

(e.g., peptides) that typically exhibit poor binding to polystyrene• Pre-blocked with your choice of Blocker™ BSA or SuperBlock®

Blocking Buffer

Reacti-Bind™ Streptavidin Coated Plate Characteristics96-Well Plate 384-Well Plate

Binding Capacity 5 pmoles/well 4 pmoles/wellCoat Volume 100 µl/well* 50 µl/wellBlocking Volume 200 µl/well 100 µl/well* Product # 15125 is coated with 200 µl of streptavidin coating solution.

ReferencesEstrada, G., et al. (1996). Mol. Cell Probes 10, 179-185.Grobler, J.A., et al. (2002). Proc. Natl. Acad. Sci. 99, 6661-6666. Hong, P. W.-P., et al. (2002). J. Virol. 76, 12855-12865. Stühlinger, M.C., et al. (2001). Circulation 104, 2569-2575. Su, S.V., et al. (2004). J. Biol. Chem. In press.


Product # Description Pkg. Size Price15120 Clear 8-Well Strip Plates with 5 plates $183

SuperBlock® Blocking Buffer15121 Clear 8-Well Strip Plates with Blocker™ BSA 5 plates $18315122 Clear 8-Well Strip Plates with 25 plates $717


SuperBlock® Blocking Buffer15125 Clear 96-Well Plates with Blocker™ BSA 5 plates $13515126 Clear 96-Well Plates with 25 plates $559






SuperBlock® Blocking Buffer15115 Reacti-Bind™ Biotin Binding Plate 4 plates $112

Sample PackOne each of the following plates: Product #s 15120, 15121, 15127 and 15128

17COATEDMICROPLATES

18

COATEDMICROPLATES

Reacti-Bind™ Streptavidin HBC Coated PlatesTake advantage of a Pierce technology that provides a broader dynamic range.

Reacti-Bind™ Streptavidin High Binding Capacity (HBC) CoatedPlates are designed for binding biotinylated oligonucleotides andpeptides with higher binding efficiency than other commerciallyavailable plates. Pierce’s proprietary coating technology (patentpending) has created a streptavidin-coated plate with four- tofive-times the binding capacity of competitors’ plates. Using aReacti-Bind™ Streptavidin HBC Plate can result in an assay witha broader dynamic range and better linearity, leading to improvedassay precision. The figure below demonstrates this effect whenmeasuring fluorescent polymerase chain reaction (PCR)products hybridized to biotinylated oligonucleotides bound toa Reacti-Bind™ High Binding Capacity (HBC) Plate and a leadingcompetitor’s high binding capacity plate (CHC). Try theReacti-Bind™ Streptavidin HBC Coated Plate and see whathas been going undetected in your research.

Figure 10. Comparison of Streptavidin High Binding Capacity (HBC) andcompetitor’s HBC coated plates. Fluoresceinated oligonucleotide hybridizationassay comparison. Comparison of Reacti-Bind™ Streptavidin High BindingCapacity (HBC) Coated Plate with competing high binding capacity plate(CHC). Plates were incubated with a biotinylated oligonucleotide, washedand probed with a complementary oligonucleotide labeled with fluoresceinat various dilutions. The Y-axis is described as the signal:noise (S/N) ratio.

Highlights:• Broader dynamic range – extends the quantitative range so

there’s no need for dilutions• Better sensitivity – increased binding capacity allows direct

detection of small ligands not observed with regular bindingcapacity plates

• Superior assay precision – standard curve demonstratesgreater linearity

• Save time – pre-blocked to reduce number of assay steps• Flexible assay formats – offered in 96- and 384-well formats

and in different colors

Reacti-Bind™ Streptavidin HBC Coated Plate Characteristics96-Well Plate 384-Well Plate


ReferenceWilson, D.S., et al. (2001). Proc. Natl. Acad. Sci. 98, 3750-3755.


Product # Description Pkg. Size Price15500 Clear 96-Well Plates with SuperBlock® 5 plates $146

Blocking Buffer*15501 Clear 8-Well Strip Plates with SuperBlock® 5 plates $194



Blocking Buffer*15504 Clear 384-Well Plates with SuperBlock® 5 plates $298



Blocking Buffer** U.S. patent pending on high-binding capacity plate-coating technology.

S/N

Ratio

Fluoresceinated Oligonucleotide (pM/well)

CHCHBC

0 5 10 20 2515 30

180

160

140

120

100

80

60

40

20

0


19COATEDMICROPLATES

HisGrab™ Nickel Coated PlatesBind recombinantly expressed fusion proteins containing a histidine tag for easy ELISA analysis.

These Ni2+ chelate-coated plates provide a simple format forprotein:protein interaction studies.

Figure 11. Binding comparison of histidine-tagged ATF fusion protein.

References1. Desai, S., et al. (1997). Previews 1(4), 12-15.Pan, W., et al. (2003). J. Biol. Chem. 278, 27820-27827. Tu, Y., et al. (1999). Mol. Cell. Biol. 19, 2425-2434.

Highlights:• Detergents used to lyse cells do not inhibit binding to Ni2+-

coated plates as they do with plain polystyrene• The detection limit is 1 ng of histidine fusion protein• Better binding for more sensitive assays compared to other

commercially available nickel-activated plates1

• Can be custom-made with other metals. Contact PierceBulk & Custom Sales or your local distributor

• Special blocking solution (Product # 37547) improvessignal:noise ratio for enhanced assay sensitivity


Product # Description Pkg. Size Price15142 Clear 8-Well Strip Plates 5 plates $16815242 White 96-Well Plates 5 plates $16815342 Black 96-Well Plates 5 plates $168

Related Pierce Products:

U.S.Product # Description Pkg. Size Price37547 Blocker™ Metal Chelate 20 ml $ 32

Compatible Formulation

Abso

rban

ce a

t 450

nm

ng ATF

Brand QPierce

0 1.15 3.125 6.25 12.5 25 50 100

1.0

0.8

0.6

0.4

0.2

0

20

COATEDMICROPLATES


HisGrab™ Copper Coated High Binding Capacity (HBC) PlatesBoost assay performance by binding more histidine-tagged protein.

HisGrab™ Copper Coated High Binding Capacity (HBC) Platesuse an exclusive coating process to increase the amount ofhistidine-tagged protein that will bind to the plate surface. Thisis ideal for high-throughput screening applications that needimproved sensitivity and greater dynamic range.

Figure 12. Binding comparison of a histidine-tagged fluorescent fusionprotein to standard HisGrab™ Nickel Coated and Copper Coated HighBinding Capacity (HBC) Plates. HisGrab™ Copper Coated HBC Plates exhibita four-fold greater capacity for binding purified polyhistidine-tagged proteinwhen assayed using a 100 µl volume. Incubation time was two hours for thebinding of polyhistidine-tagged fusion protein.

Highlights:• Quantitate previously undetectable histidine-tagged proteins• Wider dynamic range with four-fold greater capacity than

regular nickel chelate-coated plates• Special blocking solution (Product # 37547) improves

signal:noise ratio for enhanced assay sensitivity• Copper chelate provides greater binding capacity


Product # Description Pkg. Size Price15143 Clear 96-Well Plates* 5 plates $18615146 Clear 8-Well Strip Plates* 5 plates $19715147 White 96-Well Plates* 5 plates $18615148 Black 96-Well Plates* 5 plates $186* U.S. patent pending on high-binding capacity plate-coating technology.

Related Pierce Products:

U.S.Product # Description Pkg. Size Price37547 Blocker™ Metal Chelate 20 ml $ 32

Compatible Formulation

Boun

d RF

U

PHT Fusion Protein Applied, Units

8.9 pmoles

36.5 pmoles

Nickel CoatedHBC Copper Coated

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

60,000

50,000

40,000

30,000

20,000

10,000

0

21COATEDMICROPLATES

Reacti-Bind™ Anti-GST Coated PlatesA unique alternative to glutathione-coated plates for binding GST fusion proteins.

Figure 13. Reacti-Bind™ Anti-GST Coated Plates were tested for theirability to bind and detect GST. The plates were assayed with purified GSTthat was detected using a rabbit anti-GST antibody (1 mg/ml) followed bydonkey anti-rabbit HRP conjugate. The signal was developed with 1-Step™

Turbo TMB Substrate (Product # 34022).

Anti-GST antibodies often provide better binding of GST fusionproteins because protein expressing GST may not fold properlyor it may become denatured during the extraction process.This could result in poor binding to the glutathione ligand.

Highlights:• Lower detection limit is 7.5 ng of GST• Binds native or denatured forms of GST; an alternative to

glutathione-coated plates• Pre-blocked with SuperBlock® Buffer• Poppers™ Cell Lysis Reagents will not interfere with GST

fusion protein binding


Product # Description Pkg. Size Price15145 Clear 8-Well Strip Plates 5 plates $141

Abso

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t 450

nm

Purified GST Conc. (ng/ml)

Anti-GST (Pierce)

0 10 1,000

1.0

0.8

0.6

0.4

0.2

0

Reacti-Bind™ Glutathione Coated PlatesBind fusion proteins containing GST.

These plates provide easy quantitation of antibodies raisedagainst GST-fused proteins.

Highlights:• The lower detection limit is 1 ng of GST fusion protein• Simple format for protein:protein interaction studies• Detergents used to lyse cells don’t inhibit binding to

precoated plates as they do with plain polystyrene• Pre-blocked with SuperBlock® Buffer to reduce

nonspecific binding

ReferencesMcKevitt, M., et al. (2003). Genome Res. 13, 1665-1674. Pullen, S.S., et al. (1999). J. Biol. Chem. 274, 14246-14254. Yarwood, S.J., et al. (1999). J. Biol. Chem. 274, 14909-14917. Ordering Information

U.S.Product # Description Pkg. Size Price15140 Clear 8-Well Strip Plates 5 plates $14115240 White 96-Well Plates 5 plates $14115340 Black 96-Well Plates 5 plates $141

Abso

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t 450

nm

Amount of GST applied to each well (ng)

GST bound to non-coated,pre-blocked plate

GST bound to PierceGlutathione Coated Plate

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

0.8

0.6

0.4

0.2

0.0

CellScreen™ Coated PlatesOur coated plate product line is growing … cells.

The plate-coating experts at Pierce have developed CellScreen™

Coated Plates, a line of plates for cell-based assays. Thesecoated plates enable firm attachment and efficient growth ofcells for cell-based applications such as reporter assays, receptorbinding assays, cytotoxicity assays, apoptosis assays and cellproliferation assays.

The CellScreen™ Product Line includes Poly-D-Lysine andCollagen I coatings in 96- and 384-well plate formats. CellScreen™

Plates are produced using high-quality raw materials andautomation-compatible plates and lids for your high-throughputapplications. Following Pierce’s proprietary coating procedure,all CellScreen™ Plates are treated with a predetermined radiationdose. This validated irradiation process provides a moreconsistently coated plate product for cell-based assays.

Pierce has extensive microplate-coating capabilities usingmanufacturing and quality assurance equipment designed forefficient production and testing of coated microplates in largequantities. This results in economical pricing for customers.

Highlights:• High-quality base plate and lid designed for automated systems• Post-treated using a validated irradiation process• Produced using automated production and quality analysis

equipment• Reliable for use in high-throughput screening equipment

and robotics• Highly controlled procedures result in superior lot-to-lot

consistency and assurance of quality• Economical for high-volume use – contact Pierce for a bulk quote• Large lot size capability, consistency in production and

quality assurance

CellScreen™ Coated Plate Characteristics96-Well 384-Well

Plate Type Polystyrene PolystyrenePlate Bottom Flat FlatWell Volume 300 µl 100 µlGrowth Surface Area 3.3 mm2/well 1.6 mm2/wellLid Included, contains Included

condensation ringsPlate Colors Available Clear and black Clear and black

with clear bottom with clear bottom

CellScreen™ Poly-D-Lysine Coated 96- and 384-Well Plates*


Product # Description Pkg. Size Price15600 Clear 96-Well Plates 5 plates $ 5115608 Black with Clear Bottom 384-Well Plates 5 plates $122

CellScreen™ Collagen I Coated 96- and 384-Well Plates*


Product # Description Pkg. Size Price15610 Clear 96-Well Plates 5 plates $ 5115618 Black with Clear Bottom 384-Well Plates 5 plates $122

* All CellScreen™ Coated Plates are available at a special pricein bulk quantities of 200 or more plates.

Interested in another base-plate color or coating?In addition to our catalog offering, the Pierce ChoiceCoat®

Custom Plate-Coating Service can custom-coat Poly-D-Lysine,Poly-L-Lysine or Collagen on 96- or 384-well plates. ContactBulk & Custom Sales for information regarding quantity,availability and pricing.

22

COATEDMICROPLATES


Reacti-Bind™ Maleic Anhydride PlatesProteins and other primary amine-containing compounds covalently attach to the microplate.

Great for immobilization of compounds that do not normallystick to plain polystyrene plates.

Highlights:• Spontaneously react with primary amines• Maleic anhydride retains its integrity and coupling availability

for months

ReferencesBatista, F.D. and Neuberger, M.S. (2000). EMBO J. 19, 513-520. Brett, P.J., et al. (2002). J. Biol. Chem. 277, 20468-20476.Dumoutier, L., et al. (2001). J. Immunol. 166, 7090-7095.Leu, S.-J., et al. (2003). J. Biol. Chem. 278, 33801-33808. Liao, Y.-F., et al. (2002). J. Biol. Chem. 277, 14467-14474.Marston, E.L., et al. (2002). Clin. Diagn. Lab. Immunol. 9, 446-452.


Product # Description Pkg. Size Price15110 Clear 96-Well Plates 5 plates $ 6715112 Clear 96-Well Plates 25 plates $30915100 Clear 8-Well Strip Plates 5 plates $ 9015102 Clear 8-Well Strip Plates 25 plates $359

Reacti-Bind™ Maleimide Activated PlatesA convenient alternative to amine-reactive chemistries for attaching sulfhydryl-containing compounds.

Maleimide groups specifically and covalently conjugatesulfhydryl groups at neutral pH, creating a stable thioether bond.

Highlights:• Pre-blocked to reduce nonspecific binding• Convenient 8-well strip format• Easy (spontaneous) immobilization of peptides derivatized

with a terminal cysteine and proteins with free sulfhydryl

ReferencesOstrowski, M., et al. (2002). J. Virol. 76, 4241-4250.Reinhard, M., et al. (1999). J. Biol. Chem. 274, 13410-13418.


Product # Description Pkg. Size Price15150 Clear 8-Well Strip Plates 5 plates $111

H2N —

pH ≥7.2

Reaction Scheme for Coupling both Large and Small Amine-Containing Molecules


Maleic Anhydride shown anchored to polystyrene well of a microplate.Both the well wall and base are activated.

Stable amide bond formation withamines, with a resulting hydrophilic well surface.

R

R

R

O

O

C

CHH

N

C C

O( )n

O

OOCC

( )n

CC

SH —

pH ≥ 6.5-7.5

Reaction Scheme for Coupling both Large and Small Sulfhydryl-Containing Molecules

Maleimide shown anchored to polystyrene well of a microplate.Both the well wall and base are activated.

Stable thioether bond formation with sulfhydryls, with a resulting hydrophilic well surface.


R

RR

H

N

SO

O

( )n

N

O

O

( )n

23COATEDMICROPLATES

BLOCKINGAND WASHING

Blocking Unoccupied Sites

In an ELISA, it is important to block the unoccupied sites onthe surface of the well to reduce the amount of nonspecificbinding of proteins during subsequent steps in the assay. Avariety of blocking buffers ranging from nonfat milk to highlypurified proteins have been used to block unreacted sites.The blocking buffer should improve the sensitivity of theassay by reducing the background interference. An individualblocking buffer will not be compatible with every system; forthis reason, a variety of blockers in both Tris buffered saline(TBS) and phosphate buffered saline (PBS) are available. Theproper choice of blocker for a given assay depends on theantigen itself and on the type of enzyme conjugate to be used.For example, with applications using an alkaline phosphataseconjugate, a blocking buffer in TBS should be selected becausePBS interferes with alkaline phosphatase. The ideal blockingbuffer will bind to all potential sites of nonspecific interaction,eliminating background altogether, without altering or obscuringthe epitope for antibody binding.

For true optimization of the blocking step for a particularimmunoassay, empirical testing is essential. Many factors caninfluence nonspecific binding, including various protein:proteininteractions unique to a given ELISA. The most importantparameter when selecting a blocker is the signal:noise ratio,which is measured as the signal obtained with a sample containingthe target analyte as compared to that obtained with a samplewithout the target analyte. Using inadequate amounts of blockerwill result in excessive background and a reduced signal:noiseratio. Using excessive concentrations of blocker may maskantibody-antigen interactions or inhibit the enzyme, againcausing a reduction of the signal:noise ratio. When developingany new ELISA, it is important to test several different blockersfor the highest signal:noise ratio in the assay. No single blockingagent is ideal for every occasion because each antibody-antigenpair has unique characteristics.

Pierce offers a complete line of blocking buffers for ELISAincluding StartingBlock™, SuperBlock®, Casein, BSA, SEABLOCK and BLOTTO Blocking Buffers.

• StartingBlock™ Buffer contains no biotin or serum proteinand is compatible with a wide range of detection systems.StartingBlock™ Buffer works almost immediately on ELISAplates, requiring no incubation step.

• SuperBlock® Blocking Buffer is a highly purified non-serumprotein solution that is the ideal blocking agent in many assays.The blocking ability of SuperBlock® Buffers and their abilityto maintain a high signal:noise ratio is superior to most otherformulations. SuperBlock® Blocking Buffers also accomplishblocking very quickly, often in as little as 10 minutes.

• Blocker™ Casein is a 1% (w/v) ready-to-use solution ofHammersten Grade casein that can be used to blocknonspecific sites. Since casein is a purified protein, it is lesslikely than BLOTTO or other complex protein mixtures tocross-react with the antibodies and cause high background.

• Blocker™ BSA is a 10% solution of high-quality bovine serumalbumin (BSA). BSA is a commonly used blocking agent forall immunoassay applications. Blocker™ BSA is concentratedand must be diluted before use. One to three percent BSAsolutions are commonly used for blocking nonspecific sites.

• SEA BLOCK Blocking Buffer is made from steelhead salmonserum. As a non-mammalian protein blocker, the risk ofbackground caused by nonspecific interactions is minimized.

• Blocker™ BLOTTO is a ready-to-use blocking buffer madefrom nonfat dry milk. Nonfat dry milk contains endogenousbiotin and should not be used with avidin-biotin systems.

24 800-874-3723 • 815-968-0747 • www.piercenet.com

StartingBlock™ Blocking BufferStartingBlock™ Blocking Buffer simplifies the selection of a blocker for ELISA applications.

Although no blockingbuffer is ideal for everysystem, you can improvethe odds dramaticallywith StartingBlock™

Blocking Buffer becauseit is compatible with the widest variety of antibodies.

For example: StartingBlock™ Blocking Buffers are compatiblewith biotin-containing systems, while milk-based protein blockersinterfere. StartingBlock™ Buffers do not cross-react with rabbitantibodies, while many other blockers do. StartingBlock™ BlockingBuffers are also free of potentially interfering serum proteins.

StartingBlock™ Blocking Buffers offer a high level of performance –regardless of the system you choose for your ELISA or Westernblotting application. In fact, they may be the only blockers youever use.

Highlights:Compatible with a wide range of detection systems• Works in both ELISA and Western applications• Does not cross-react with rabbit antibodies • Serum protein-free• Biotin-free

Shorter blocking times• “No-wait” blocking capability

Superior signal:noise ratios in ELISA applications• Signal:noise ratios in the range of 10:1-20:1 have been realized

with StartingBlock™ Blocking Buffer


Product # Description Pkg. Size Price37538 StartingBlock™ (PBS) Blocking Buffer 1 liter $127

A protein-based blocker formulation in phosphate buffered saline (pH 7.5) for use in ELISA and Western blotting applications.

37542 StartingBlock™ (TBS) Blocking Buffer 1 liter $127A protein-based blocker formulation in Tris buffered saline (pH 7.5) for use in ELISA and Western blotting applications.

Starting Block™ Blocking Buffers are also available with anoptimized amount of Tween®-20 Detergent to provide the lowest background.


Product # Description Pkg. Size Price37539 StartingBlock™ T20 (PBS) Blocking Buffer 1 liter $139

A protein-based blocker formulation in phosphate buffered saline at pH 7.5 with 0.05% Tween®-20 and Kathon® Antimicrobial Agent.

37543 StartingBlock™ T20 (TBS) Blocking Buffer 1 liter $139A protein-based blocker formulation in Tris buffered saline at pH 7.5 with 0.05% Tween®-20 and Kathon® Antimicrobial Agent.

25BLOCKINGAND WASHING

SEA BLOCK Blocking BufferNo mammalian proteins, reducing the risk of nonspecific interaction.

Highlights:• Made from steelhead salmon serum• Functions as a universal blocker• Offers reduced background• Can be diluted up to 1:10 with buffer

ReferencesHypolite, J.A., et al. (2001). Amer. J. Physiol. – Cell. Physiol. 280, C254-264. Wang, L., et al. (2002). J. Clin. Invest. 110, 1175-1184.


Product # Description Pkg. Size Price37527 SEA BLOCK Blocking Buffer 500 ml $121

SuperBlock® Dry Blend (TBS) Blocking BufferDelivers the ultimate in space-saving convenience.

Highlights:• Delivers even more economy and stability• Each pouch reconstitutes to form 200 ml of SuperBlock®

Blocking Buffer in TBS• Room-temperature storage; small packaging takes up

minimal shelf space

ReferencesIkeda, K., et al. (2003). J. Biol. Chem. 278, 7725-7734.Leclerc, G.J. and Barredo, J.C. (2001). Clin. Cancer Res. 7, 942-951.Subbarayan, V., et al. (2001). Cancer Res. 61, 2720-276. Walters, R.W., et al. (2002). Cell 100, 789-799.


Product # Description Pkg. Size Price37545 SuperBlock® (TBS) Blocking Buffer 5 pouches $ 98

Dry Blend Blocking BufferEach pouch yields 200 ml when reconstituted.

26

BLOCKINGAND WASHINGSuperBlock® Blocking BuffersGuaranteed to be biotin-free.

Our most popular blocking buffer, SuperBlock® BlockingBuffer, now comes in both dry and liquid formats! Manyresearchers have discovered that SuperBlock® Blocking Bufferis the only blocking buffer needed for all of their applications.

Highlights:• Fast blocking – blocks ELISA plates in two minutes or

membranes in five to 10 minutes• Non-serum protein solution yields a very high signal:noise ratio• Plates blocked with SuperBlock® Blocking Buffer can be

stored dry for up to 12 months• Liquid formulations available in PBS or TBS• Biotin-free


Product # Description Pkg. Size Price37515 SuperBlock® (PBS) Blocking Buffer 1 liter $11837535 SuperBlock® (TBS) Blocking Buffer 1 liter $118

SuperBlock® Blocking Buffers are also available with an optimizedamount of Tween®-20 Detergent to provide the lowest background.


Product # Description Pkg. Size Price37516 SuperBlock® T20 (PBS) Blocking Buffer 1 liter $129

(Contains 0.05% Tween®-20)37536 SuperBlock® T20 (TBS) Blocking Buffer 1 liter $129

(Contains 0.05% Tween®-20)


BLOCKINGAND WASHING

Blocker™ BSAFor all blocking applications.

Highlights:• 10% solutions of high-quality bovine serum albumin• Concentrated formulation saves storage space• No waiting for powder to dissolve with this ready-to-dilute

liquid concentrate


Product # Description Pkg. Size Price37525 Blocker™ BSA in PBS (10X) 200 ml $10237520 Blocker™ BSA in TBS (10X) 125 ml $ 98

Blocker™ BLOTTOReady-to-use blocking buffers made of nonfat dry milk.

Highlights:• Preformulated for ease of use• Anti-foaming agent added• Available in TBS Buffer• Merthiolate-free formulation

ReferencesGoretzki, L., et al. (2000). J. Biol. Chem. 275, 28625-28633. Abrams, E.T., et al. (2003). J. Immunol. 170, 2759-2764.


Product # Description Pkg. Size Price37530 Blocker™ BLOTTO in TBS 1 liter $ 79

5% (w/v) nonfat powdered milk in TBS, 0.01%Anti-foam A, contains Kathon® AntimicrobialReagent as preservative, pH 7.4.

Blocker™ CaseinReady-to-use solution (1% w/v) of Hammersten Grade casein for blocking nonspecific sites.

Highlights:• Preformulated for ease of use• Use when skim milk demonstrates high background problems• Thimerosal-free formulation

ReferencesNemzek, J.A., et al. (2000). Amer. J. Physiol. – Lung Cell. Mol. Physiol. 278, L512-520. Stuyver, L.J., et al. (2003). Antimicrob. Agents Chemother. 47, 244-254. Wolfman, J.C., et al. (2002). Mol. Cell. Biol. 22, 1589-1606.


Product # Description Pkg. Size Price37532 Blocker™ Casein in TBS 1 liter $ 83

1% (w/v) Casein Hammersten Grade in TBS, Contains Kathon® Antimicrobial Reagent as preservative, pH 7.4.

37528 Blocker™ Casein in PBS 1 liter $ 831% (w/v) Casein Hammersten Grade in PBS, Contains Kathon® Antimicrobial Reagent as preservative, pH 7.4.

27

28

BLOCKINGAND WASHING

Blocking Buffers Application Chart

Immunohisto- DNA/RNA U.S.Product # Blocking Buffer ELISA Western blot Dot Blot chemistry Hybridizations Price37538 StartingBlock™ (PBS) Blocking Buffer ✔ ✔ ✔ ✔ $12737542 StartingBlock™ (TBS) Blocking Buffer ✔ ✔ ✔ ✔ $12737539 StartingBlock™ T20 (PBS) Blocking Buffer ✔ ✔ ✔ ✔ $13937543 StartingBlock™ T20 (TBS) Blocking Buffer ✔ ✔ ✔ ✔ $13937515 SuperBlock® Blocking Buffer in PBS ✔ ✔ ✔ ✔ ✔ $11837535 SuperBlock® Blocking Buffer in TBS ✔ ✔ ✔ ✔ ✔ $11837516 SuperBlock® T-20 PBS Blocking Buffer ✔ ✔ ✔ ✔ ✔ $12937536 SuperBlock® T-20 TBS Blocking Buffer ✔ ✔ ✔ ✔ ✔ $12937527 SEA BLOCK Blocking Buffer ✔ ✔ ✔ $12137520 Blocker™ BSA in TBS ✔ ✔ ✔ ✔ ✔ $ 9837525 Blocker™ BSA in PBS ✔ ✔ ✔ ✔ ✔ $10237532 Blocker™ Casein in TBS ✔ ✔ ✔ ✔ ✔ $ 8337528 Blocker™ Casein in PBS ✔ ✔ ✔ ✔ ✔ $ 8337530 Blocker™ BLOTTO in TBS ✔ ✔ ✔ ✔ ✔ $ 7937547 Blocker™ Metal Chelate ✔ $ 32

Blocker™ Metal Chelate Compatible FormulationDesigned to improve assay sensitivity for metal chelate-based assay systems.

Highlights:• Improves assay sensitivity when using HisGrab™ Metal

Chelate High Binding Capacity (HBC) and regular HisGrab™

Metal Chelate Coated plates• Use at a 1:1,000 dilution to dilute assay reagents for best

signal:noise ratio• Thimerosal-free formulation


Product # Description Pkg. Size Price37547 Blocker™ Metal Chelate Compatible 20 ml $ 32

FormulationUse concentrate at 1:1,000 dilution, pH 7.4.

Surfact-Amps® 20 Purified Detergent Solution Specially purified form of Tween®-20 Detergent.

Highlights:• Guaranteed < 1 milliequivalent of peroxides and carbonyl in a

10% solution• Enhances signal:background ratio

ReferenceEbong, S.J., et al. (2001). Infect. Immun. 69, 2099-2106.


Product # Description Pkg. Size Price28320 Surfact-Amps® 20 Purified 6 x 10 ml $ 82

Detergent Solution


BLOCKINGAND WASHING

Surfact-Amps® Purified Detergent SolutionsNothing could be easier … nothing protects better!

Highlights:• Disrupts nonspecific

protein interactions• Ampules are packed

under nitrogen formaximum shelf life and are pre-scored foreasy opening

• Gentle – non-denaturingat low concentrations

• Clear and pure 10% solutions – less than 1.0 µeq/ml peroxidesand carbonyl content

• Unparalleled convenience – solutions are purified, predilutedand ampuled


Product # Description Pkg. Size Price

Tween®-20-Based Detergents

28320 Surfact-Amps® 20 6 x 10 ml $ 82(Active Ingredient: Tween®-20)

28328 Surfact-Amps® 80 6 x 10 ml $ 82(Active Ingredient: Tween®-80)

Triton®-based Detergents

28314 Surfact-Amps® X-100 6 x 10 ml $ 82(Active Ingredient: Triton® X-100)

28332 Surfact-Amps® X-114 6 x 10 ml $192(Active Ingredient: Triton® X-114)

Nonidet-based Detergent

28324 Surfact-Amps® NP-40 6 x 10 ml $ 82(Active Ingredient: Nonidet P-40)

Brij®-based Detergents

28316 Surfact-Amps® 35 6 x 10 ml $ 82(Active Ingredient: Brij®-35)

28336 Surfact-Amps® 58 6 x 10 ml $119(Active Ingredient: Brij®-58)

20150 Brij® -35, 30% Solution 950 ml $ 75Active ingredient is supplied as a purified 10% aqueous solution ampuled under nitrogen.

29

Surfact-Pak™ Detergent SamplerConvenient 10-sample package of detergents allows for testing and experimentation.


Product # Description Pkg. Size Price28340 Surfact-Pak™ Detergent Sampler Kit $239

Contains: Surfact-Amps® Purified DetergentsSurfact-Amps® X-100 10 mlSurfact-Amps® 35 10 mlSurfact-Amps® 20 10 mlSurfact-Amps® NP-40 10 mlSurfact-Amps® 80 10 mlSurfact-Amps® X-114 10 mlSurfact-Amps® 58 10 ml

Octyl β-Glucoside 100 mgOctyl β-Thioglucopyranoside 100 mgCHAPS 100 mg

30

BLOCKINGAND WASHING

Washing the Microplate

Many immunoassay procedures include a series of incubationswith different immunochemical reagents separated by wash steps.Washing steps are necessary to remove unbound reagentsand reduce background, thereby increasing the signal:noiseratio. Insufficient washing will allow high background, whileexcessive washing may result in decreased sensitivity causedby elution of the antibody and/or antigen from the well. A varietyof buffers may be used. Occasionally, washing is performed ina physiologic buffer such as Tris buffered saline (TBS) orphosphate buffered saline (PBS) without any additives.More commonly, a detergent such as 0.05% Tween®-20(Product # 28320) is added to the buffer to help removenonspecifically bound material. Another common techniqueis to use a dilute solution of the blocking buffer along withsome added detergent. Including the blocking agent and addinga detergent in wash buffers helps to minimize background inthe assay. For best results, use high-purity detergents, suchas Surfact-Amps® Detergents.

BupH™ Dry BuffersThe most advanced, versatile, time-saving buffer product line available.The ultimate in convenience1. Reach for the sealed foil pack sitting conveniently on your

bench top.2. Open, pour into beaker and add water.3. The fresh buffer is ready to use in practical aliquots so

there’s no waste.The ultimate in versatility1. Routine buffers are designed for use in Western blotting,

dialysis, cross-linking, ELISAs, immunohistochemistry,protein plate-coating, biotinylation and other applications.

2. Using one buffer source maintains consistency and eliminatesvariables within the lab.

The ultimate in integrity1. BupH™ Buffers are protected from contamination and are

fresh every time. 2. Carry out applications with confidence in buffer quality.3. “Test-assured” with the Pierce commitment to quality

management standards.The ultimate in time savings1. Making routine buffers is no longer time-consuming.2. No component measurement, pH adjustment, quality validation,

preparation tracking or refrigeration hassles.3. Move forward with your work by eliminating re-tests due to

buffer problems.

CHAPS & CHAPSOZwitterionic detergents that are ideal for protecting the native state of proteins.

Highlights:• Non-denaturing• Able to disrupt nonspecific protein interactions• Less protein aggregation than nonionic detergents• Electrically neutral


Product # Description Pkg. Size Price28300 CHAPS 5 g $ 99

(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)

28299 CHAPS 100 g $82928304 CHAPSO 5 g $209

(3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate)

HO OH

OH O

N+

HO SO

O

O–

CHAPSM.W. 614.88

CHAPSOM.W. 630.88

NH


BLOCKINGAND WASHING 31

Surfact-Amps® 20 Purified Detergent Solution Specially purified form of Tween ®-20.

Highlights:• Can be added to PBS or TBS wash buffers to improve performance• Guaranteed < 1 milliequivalent of peroxides and carbonyl in a

10% solution• Enhances signal:background ratio


Product # Description Pkg. Size Price28320 Surfact-Amps® 20 6 x 10 ml $ 82

BupH™ Tris Buffered SalineGreat wash buffer!

Each pack yields 500 ml of 25 mM Tris, 0.15 M NaCl, pH 7.2when dissolved in 500 ml deionized water (10 pack makes5 liters total; 40 pack makes 20 liters total).


Product # Description Pkg. Size Price28380 BupH™ Tris-Glycine Buffer Packs 40 pack $ 9828376 BupH™ Tris Buffered Saline Packs 40 pack $10928379 BupH™ Tris Buffered Saline Packs 10 pack $ 49

BupH™ Phosphate Buffered Saline PacksGreat wash buffer!

Each pack yields 500 ml of 0.1 M phosphate, 0.15 M NaCl, pH7.0 when dissolved in 500 ml deionized water (20 liters total).


Product # Description Pkg. Size Price28372 BupH™ Phosphate Buffered Saline Packs 40 pack $109

Affinity-Purified Primary Antibodies

The choice of a primary antibody will depend on the antigen to bedetected and what antibodies are available to that antigen. Ahuge number of primary antibodies are available commerciallyand can be identified quickly by searching sites such aswww.antibodyresource.com on the Internet. Alternatively, aprimary antibody may be made to recognize the antigen ofinterest. For more information on producing a custom antibody,see the Antibody Production and Purification technical sectionof the Pierce Technical Handbook and Catalog. Both polyclonaland monoclonal antibodies work well for ELISA. Polyclonalantibodies are less expensive and less time-consuming to produceand they often have a high affinity for the antigen. Monoclonalantibodies are valued for their specificity, purity and consistencythat result in lower background. Crude antibody preparationssuch as serum or ascites fluid are sometimes used for ELISA,but the impurities present may increase background. To obtainantibodies with the greatest specificity, they can be affinity-purified using the immobilized antigen. For more informationon affinity purification, order your FREE Affinity PurificationHandbook from a Pierce Customer Service representative at800-874-3723 or 815-968-0747, or from your local PerbioBranch office or distributor.

A wide variety of labeled secondary antibodies can be used forELISA detection. The choice of secondary antibody dependsupon the species of animal in which the primary antibody wasraised (the host species). For example, if the primary antibodyis a mouse monoclonal antibody, the secondary antibody mustbe an anti-mouse antibody obtained from a host other than themouse. The host species of the secondary antibody often willnot affect the experiment. However, secondary antibodies areavailable from several different host species and, if a secondaryantibody causes high background in a particular assay, another

host species may be chosen. Another option to reduce backgroundis to use a secondary antibody that has been pre-adsorbed toserum proteins from other species. This pre-adsorption processremoves antibodies that have the potential to cross-react withserum proteins, including antibodies, from those species. Toexpedite the process of choosing the appropriate secondaryantibody, visit the Secondary Antibody Selection Guide locatedunder the Products tab on the Pierce web site.

Antibodies for ELISA are typically used as dilute solutions,ranging from 1/100-1/500,000 dilutions beginning from a 1 mg/ml stock solution. The optimal dilution of a given antibodywith a particular detection system must be determinedexperimentally. More sensitive detection systems require thatless antibody be used, which can result in substantial savingson antibody costs and allow a limited supply of antibody tobe stretched out over more experiments. It also produces aside benefit of reduced background because the limited amountof antibody shows increased specificity for the target with thehighest affinity. Antibody dilutions are typically made in thewash buffer containing a blocking agent. The presence of asmall amount of blocking agent and detergent in the antibodydiluent often helps to minimize background.

Pierce offers a wide variety of ImmunoPure® Labeled SecondaryAntibodies for use in ELISA. The labels include biotin, fluorescein,rhodamine, horseradish peroxidase and alkaline phosphatase.For the complete list of labeled secondary antibodies pleaserefer to pages 36-38.

32

PRIMARY AND SECONDARY ANTIBODIES


Antibody LabelsThe choice of secondary antibody also depends upon the typeof label that is desired. Many different labels can be conjugatedto antibodies. Radioisotopes were used extensively in thepast, but they are expensive, have a short shelf life, offer noimprovement in signal:noise ratio and require special handling.Alternative labels are biotin, fluorophores and enzymes. The useof fluorophores requires fewer steps; however, special equipmentis needed to view the fluorescence. Also, a photograph must betaken if a permanent record of the results is desired. Enzymaticlabels are used most commonly and, although they requireextra steps, they can also be extremely sensitive.

Alkaline phosphatase (AP) and horseradish peroxidase (HRP) arethe two enzymes that are used extensively as labels for proteindetection. An array of chromogenic, fluorogenic and chemilu-minescent substrates is available for use with either enzyme.For a detailed comparison of these two enzymes, see Table 3.

AP, a 140 kDa protein that is generally isolated from calfintestine, catalyzes the hydrolysis of phosphate groups froma substrate molecule, resulting in a colored or fluorescentproduct or the release of light as a byproduct. AP has optimalenzymatic activity at a basic pH (pH 8-10) and can be inhibitedby cyanides, arsenate, inorganic phosphate and divalent cationchelators, such as EDTA. As a label for ELISA, AP offers adistinct advantage over other enzymes. Because its reactionrate remains linear, detection sensitivity can be improved bysimply allowing a reaction to proceed for a longer time period.

HRP is a 40 kDa protein that catalyzes the oxidation of substratesby hydrogen peroxide, resulting in a colored or fluorescentproduct or the release of light as a byproduct. HRP functionsoptimally at a near-neutral pH and can be inhibited by cyanides,sulfides and azides. Antibody-HRP conjugates are superior toantibody-AP conjugates with respect to the specific activitiesof both the enzyme and antibody. In addition, its high turnoverrate, good stability, low cost and wide availability of substratesmake HRP the enzyme of choice for most applications.

Table 3. Comparison of horseradish peroxidase and alkaline phosphatase enzymes.

Horseradish Peroxidase Alkaline PhosphataseSize 40 kDa 140 kDaPrice Relatively Inexpensive Relatively ExpensiveStability (Storage) Stable at < 0˚C Unstable at < 0˚CNumber of Substrates Many FewKinetics Rapid SlowerpH optimum 5-7 8-10

Antibody Conjugates and Enzyme Detection

After blocking, the primary antibody is incubated in themicroplate, allowing it to bind to the antigen. The time requiredfor this step typically ranges from 1 hour at room temperatureto overnight at 4°C, depending upon the affinity of the antibodyfor its antigen. The primary antibody may have been conjugated(labeled) with an enzyme or biotin molecule to create a directELISA. Care must be taken that the antibody conjugate is storedproperly and that it has been diluted in the blocking bufferwith or without Tween®-20 Detergent when performing theELISA. Peroxidase-conjugated antibodies may be stored inGuardian® Peroxidase Conjugate Stabilizer (Product # 37548)to a 1:1,000 dilution or to the desired assay dilution range.This stabilizer prevents the loss of enzyme activity over time,removes the need to aliquot and freeze conjugates, and allowsthe antibody conjugate to be stored at 4°C.

An alternative to the direct method is to use a labeled secondaryantibody that recognizes the primary antibody species. Forexample, a goat anti-mouse secondary antibody will bind toa mouse monoclonal primary antibody. Many secondaryantibody conjugates are commercially available with commonenzyme labels such as AP or HRP. If the primary antibody has abiotin tag, then the secondary reagent would be a streptavidin orNeutrAvidin™ Enzyme Conjugate, with an alternative being theavidin-biotin complex (ABC) with a biotinylated enzyme (Figure 1,page 5). Selecting the appropriate protocol for an ELISA dependsupon the availability of the primary antibody, the sensitivitylevel desired and the nature of the sample being tested. Pierceprovides a variety of secondary antibodies, NeutrAvidin™ Proteinand streptavidin conjugates to choose from in designing anELISA with low background and high reproducibility.

PRIMARY & SECONDARY ANTIBODIES 33

Table 4. Choosing a detection method.

Colorimetric Substrates Chemifluorescent Substrates Chemiluminescent SubstratesELISA • Medium/low sensitivity • High sensitivity • High sensitivity

• Generally less expensive • Generally more expensive • Generally more expensive• Many substrates available • Few substrates available • Few substrates available• Slow signal generation • Rapid signal generation • Rapid signal generation• Enzyme catalyzed quickly • Enzyme activity maintained • Enzyme catalyzed quickly• Small linear range/poor low-end linearity • Large linear range/enhanced low-end linearity • Large linear range/enhanced • Flexible (stopped, nonstopped • Flexible (stopped, nonstopped low-end linearityand kinetic assays) and kinetic assays) • Nonflexible

Detection Spectrophotometer provides Fluorometer provides Luminometer providesMethod quantifiable results quantifiable results quantifiable results

34



Enzyme-antibody conjugates often yield best results when dilutedin the same material that was used for blocking with the additionof high-quality Tween®-20 Detergent to a concentration of 0.05%.Figure 14 compares SuperBlock® Blocking Buffer with Tween®-20Detergent to other proteins for use as conjugate diluents. It hasbeen demonstrated that Tween®-20 Detergent, as a componentof the primary and secondary antibody diluents, often providesincreased signal:noise ratios. Pierce Surfact-Amps® 20 (Product #28320) is a highly purified Tween®-20 Detergent solution, freeof peroxides and carbonyls that can cause ELISA artifacts.

Figure 14. SuperBlock® Blocking Buffers vs. other proteins for use asconjugate diluents. SuperBlock® Blocking Buffers with 0.05% Tween®-20Detergent is the superior diluent.

The final stage in all ELISA systems is a detection step in whicha substrate is introduced, and the bound enzyme (indicating thepresence of analyte) converts that substrate to a detectableproduct. The intensity of the signal produced when the substrateis added should correlate to the concentration of the primaryantibody and the respective antigen. Enzyme-labeled reagentsmay be detected using chromogenic, chemifluorescent orchemiluminescent substrates. When performing ELISAs, asoluble substrate is used to generate a signal in solution.Chemiluminescent signals emit light that is measured by aluminometer while fluorescent substrates require an excitationfor light to be emitted and detected by a fluorometer (Table 4).

Abso

rban

ce a

t 490

nm

Background Levels

1.25

1.00

0.75

0.50

0.25

0.0SuperBlock™ Blocking

Buffer with .05%Tween®-20 Detergent

3% BSA 10% Skim Milk

35PRIMARY & SECONDARY ANTIBODIES

Affinity-Purified Secondary Antibodies

ImmunoPure® Affinity-Purified Antibodies are availableunconjugated or conjugated with biotin, alkaline phosphatase,horseradish peroxidase, fluorescein or rhodamine. F(ab′)2

fragments of antibodies to immunoglobulins are also availablein unconjugated or conjugated forms. These F(ab′)2 fragmentsof antibodies are especially useful in assays in which bindingbetween the Fc portions of antibodies and Fc receptor-bearingcells must be eliminated.

ImmunoPure® Polyclonal Antibodies are purified by immunoaffinitychromatography using antigens coupled to agarose gels.Affinity purification helps to eliminate nonspecific antibodies,resulting in an increase in sensitivity and specificity, with adecrease in background. The purification process involves anelution procedure, yielding antibodies with high avidity. Theseantibodies exhibit both maximal binding to antigens and minimalcross-reactivity to other molecules. Conjugated antibodies areaffinity-purified prior to the conjugation process.

Selected ImmunoPure® Antibodies have been further purified bypassing them through immobilized serum proteins from otherspecies. This additional processing minimizes cross-reactivitiesto other species’ serum proteins and is indicated by “min xSpecies Sr Prot.” The key to abbreviations for the individualspecies is shown in Table 5.

Table 5. Key to abbreviations for individual species.

Bv = Bovine Gu = Guinea Pig Hs = Horse Rt = RatCh = Chicken Ha = Hamster Ms = Mouse Sh = SheepGt = Goat Hn = Human Rb = Rabbit Sw = Swine

ImmunoPure® Polyclonal Conjugated Antibodies contain bovineserum albumin as a stabilizer. Table 6 lists the typical workingdilutions for the conjugated antibodies when doing ELISAs.

Table 6. Typical dilution ranges recommended for Pierce ImmunoPure®

Polyclonal Conjugated Antibodies when doing ELISAs.

Conjugate ELISAAlkaline Phosphatase 1:5,000-1:50,000Peroxidase 1:5,000-1:200,000

(for SuperSignal® ELISA Products)

Storing Enzyme ConjugatesPierce provides a variety of reagents to help preserve enzymeconjugate activity. Typically, conjugates are aliquoted in 50-100 µlincrements using purified ethylene glycol (Product # 29810)as a preservative for -20°C storage. Conjugates can maintainactivity for up to two years. An alternative to aliquoting is to useSuperFreeze™ Peroxidase Conjugate Stabilizer (Product # 31503),diluting the conjugate 1:1 in the stabilizer and storing it at -20°Cfor up to one year as a stock solution. Guardian® PeroxidaseStabilizer/Diluents (Product #s 37548 and 37552) allowperoxidase conjugates to be reconstituted and stored at 4°Cas a 1:1,000 dilution or a 1:100,000 dilution stock solution.

Conjugate Stabilizers


Product # Description Pkg. Size Price37548 Guardian® Peroxidase Conjugate 200 ml $ 73

Stabilizer/Diluent37552 Guardian® Peroxidase Conjugate 1 liter $223

Stabilizer/Diluent31503 SuperFreeze™ Peroxidase Conjugate 25 ml $ 49

Stabilizer29810 Ethylene Glycol 200 ml $ 75

(50% aqueous solution)


Ordering InformationProduct #Pkg. Size/Price

Specificity Description Host Unconj. Biotin-LC Fluorescein Rhodamine Peroxidase Alk. Phos.Anti-CHICKEN Chicken IgY (H+L) Rabbit 31104 31720 31501 31401

2 mg/$113 1.5 ml/$123 1.5 mg/$106 1.5 ml/$135Anti-GOAT Goat IgG (H+L) Donkey 31108

1.5 mg/$91Goat IgG (H+L) Mouse 31107 31730 31512 31400(min x HnMsRb Sr Prot)* 1.5 mg/$117 1 ml/$144 1 mg/$117 1 ml/$149

Goat IgG (H+L) Rabbit 31105 31732 31509 31650 31402 313002 mg/$106 1.5 mg/$112 1.5 mg/$98 1.5 mg/$99 1.5 ml/$125 1 ml/$156

Goat IgG [F(ab′)2] Rabbit 31153 31753 31553 31403 314052 mg/$122 1.5 ml/$138 1.5 mg/$116 1.5 ml/$148 1 ml/$138

Goat IgG (Fc) Rabbit 31133 31733 31533 31433 313372 mg/$114 1.5 ml/$146 1.5 mg/$116 1.5 ml/$144 1 ml/$178

Anti-GOAT F(ab′)2 Goat IgG (H+L) Rabbit 31109 31302Fragment of (min x Hn Sr Prot)* 0.5 mg/$113 0.5 ml/$162Host AntibodyAnti-HAMSTER Hamster IgG (H+L) Goat 31115 31750

1.5 mg/$93 1.5 mg/$114Hamster IgG (H+L) Rabbit 31120 31587 31652

2 mg/$77 1.5 mg/$93 1.5 mg/$90Anti-HORSE Horse IgG (H+L) Goat 31116 31760

2 mg/$78 1.5 mg/$112Anti-HUMAN Human IgG (H+L) Goat 31130 31770 31529 31656 31410 31310

2 mg/$81 1.5 mg/$112 2 mg/$87 2 mg/$94 2 ml/$116 1 ml/$144Human IgG Goat 31118Gamma Chain Specific 0.5 mg/$77

Human IgG (H+L) Goat 31119 31774 31531 31412(min x BvHsMs Sr Prot)* 1.5 mg/$84 1.5 ml/$114 1.5 mg/$82 1.5 ml/$120

Human IgG [F(ab′)2] Goat 31122 313122 mg/$101 1 ml/$170

Human IgG [F(ab′)2] Goat 31132 31414(min x BvHsMs Sr Prot)* 1.5 mg/$93 1.5 ml/$152

Human IgG (Fc) Goat 31123 31416(min x BvHsMs Sr Prot)* 1.5 mg/$93 1.5 ml/$154

Human IgM (Fc5µ) Goat 31136 31575 314152 mg/$90 2 mg/$119 2 ml/$151

Human IgM (µ) Goat 31124 317780.5 mg/$89 0.5 mg/$120

Human IgA (α) Goat 31140 31577 31417 313142 mg/$95 2 mg/$98 2 ml/$139 1 ml/$159

Human IgA + IgG Goat 31128 31782 31418 31316+ IgM (H+L) 2 mg/$109 2 ml/$144 2 ml/$139 1 ml/$159

Human Kappa Chain Goat 31129 317800.5 mg/$84 0.5 mg/$120

Human Lambda Chain Goat 311310.5 mg/$90

Human IgG (H+L) Mouse 31135 31420(min x Ms Sr Prot)* 2 mg/$110 1.5 ml/$121

*See Table 5 on page 35 for the Key to Abbreviations.

36


PRIMARY & SECONDARY ANTIBODIES


Specificity Description Host Unconj. Biotin-LC Fluorescein Rhodamine Peroxidase Alk. Phos.Anti-HUMAN Human IgG (H+L) Mouse 31137 31784continued (min x BvHsMs Sr Prot)* 1.5 mg/$104 1 ml/$128

Human IgG (H+L) Rabbit 31143 317862 mg/$99 1.5 ml/$113

Human IgG (Fc) Rabbit 31142 31789 31535 31423 313182 mg/$101 1.5 ml/$137 1.5 mg/$104 1.5 ml/$151 1 ml/$158

Anti-HUMAN Human IgG (Fc) Goat 31163F(ab′)2 Fragment 1 mg/$117of Host Antibody

Human IgG (H+L) Goat 311651 mg/$84

Human IgA + IgG Goat 31539+ IgM (H+L) 1 mg/$111

Anti-MOUSE Mouse IgA (α) Goat 31169(min x Hn Sr Prot)* 1 mg/$143

Mouse IgA + IgG Goat 31171+ IgM (H+L) 2 mg/$142

Mouse IgG (H+L) Goat 31160 31800 31569 31660 31430 313202 mg/$84 2 ml/$109 2 mg/$95 2 mg/$99 2 ml/$131 1 ml/$156

Mouse IgG (H+L) Goat 31164 31802 31541 31661 31432 31322(min x BvHnHs Sr Prot)* 1.5 mg/$79 1.5 mg/$125 1.5 mg/$101 1.5 mg/$99 1.5 ml/$128 1 ml/$161

Mouse IgG [F(ab′)2] Goat 31166 31803 31543 31436 313242 mg/$109 2 ml/$144 2 mg/$113 2 ml/$139 1 ml/$167

Mouse IgG (Fc) Goat 31168 31805 31547 31663 31437 313252 mg/$99 2 ml/$138 2 mg/$114 2 mg/$111 2 ml/$148 1 ml/$162

Mouse IgG (Fc) Goat 31170 31439 31327(min x BvHnHs Sr Prot)* 1.5 mg/$95 1.5 ml/$167 1 ml/$185

Mouse IgM (µ) Goat 31172 31804 31992 31662 31440 313262 mg/$114 0.5 mg/$114 2 mg/$139 2 mg/$138 2 ml/$168 1 ml/$207

Mouse IgM (µ) Goat 31176 31585 31664(min x BvHnHs Sr Prot)* 1.5 mg/$111 1.5 mg/$154 1.5 mg/$159

Mouse IgG + IgM Goat 31182 31807 31586 31444 31328(H+L) 2 mg/$90 2 ml/$138 1.5 mg/$128 2 ml/$135 1 ml/$167

Mouse IgG + IgM Goat 31184 31446 31330(H+L) (min x BvHnHs Sr Prot)* 1.5 mg/$110 1.5 ml/$167 1 ml/$215

Mouse IgG (H+L) Horse 31181 318061.5 mg/$89 1.5 mg/$109

Mouse IgG (H+L) Rabbit 31188 31810 31561 31665 31450 313292 mg/$104 1.5 ml/$120 1.5 mg/$87 1.5 mg/$82 1.5 ml/$132 1 ml/$139

Mouse IgG (H+L) Rabbit 31190 31812 31452 31334(min x Hn Sr Prot)* 1.5 mg/$99 1 ml/$125 1 ml/$138 0.5 ml/$157

Mouse IgG [F(ab′)2] Rabbit 31192 31811 31559 31666 31451 313312 mg/$105 1.5 ml/$157 1.5 mg/$106 1.5 mg/$106 1.5 ml/$161 1 ml/$158

Mouse IgG (Fc) Rabbit 31194 31813 31555 31455 313322 mg/$106 1.5 ml/$151 1.5 mg/$100 1.5 ml/$156 1 ml/$158

Mouse IgM (µ) Rabbit 31196 31814 31557 31456 313332 mg/$138 1.5 ml/$167 1.5 mg/$124 1.5 ml/$171 1 ml/$212

Mouse IgG + IgM Rabbit 31198 31815 31558 31457 31335(H+L) 2 mg/$124 1.5 ml/$146 1.5 mg/$117 1.5 ml/$143 1 ml/$145

Mouse IgG (H+L) Goat 31185 31565 31438(min x BvHnHs Sr Prot)* 1 mg/$118 1 mg/$140 0.5 ml/$148


37


Specificity Description Host Unconj. Biotin-LC Fluorescein Rhodamine Peroxidase Alk. Phos.Anti-MOUSE Mouse IgM (µ) Goat 31178F(ab′)2 Fragment 1 mg/$147of Host Antibody

Mouse IgM (µ) Goat 31186 31442(min x BvHnHs Sr Prot)* 1 mg/$149 0.5 ml/$166Mouse IgG + IgM Goat 31448(H+L) (min x BvHnHs Sr Prot)* 0.5 ml/$134

Anti-RABBIT Rabbit IgG (H+L) Donkey 31821 31568 31458 31345(min x BvGtHnHsMsRtSh Sr Prot)* 0.5 ml/$109 0.5 mg/$93 0.5 ml/$114 0.5 ml/$143

Rabbit IgG (H+L) Goat 31210 31820 31670 31460 313402 mg/$82 1.5 mg/$114 2 mg/$112 2 ml/$135 1 ml/$157

Rabbit IgG (H+L) Goat 31212 31822 31583 31462 31342(min x Hn Sr Prot)* 1.5 mg/$94 1.5 ml/$114 1.5 mg/$106 1.5 ml/$161 1 ml/$152

Rabbit IgG [F(ab′)2] Goat 31234 31823 31573 31461 313432 mg/$82 2 ml/$121 2 mg/$114 2 ml/$148 1 ml/$159

Rabbit IgG (Fc) Goat 31216 31463 313412 mg/$94 2 ml/$149 1 ml/$156

Rabbit IgG (H+L) Mouse 31213 31824 31584 31674 31464(min x GtHnMsSh Sr Prot)* 1.5 mg/$115 1 ml/$138 1 mg/$117 1 mg/$114 1 ml/$148

Anti-RABBIT Rabbit IgG (H+L) Goat 31214 31579F(ab′)2 Fragment 1 mg/$88 1 mg/$120of Host Antibody

Rabbit IgG (Fc) Goat 31217 315811 mg/$132 1 mg/$143

Anti-RAT Rat IgG (H+L) Goat 31220 31830 31629 31680 31470 313502 mg/$94 2 ml/$109 2 mg/$94 2 mg/$94 2 ml/$112 1 ml/$137

Rat IgG [F(ab′)2] Goat 314742 ml/$128

Rat IgG (Fc) Goat 31226 31833 31621 31475 313532 mg/$92 2 ml/$125 2 mg/$102 2 ml/$137 1 ml/$146

Rat IgM (µ) Goat 31228 31832 31631 31476 313542 mg/$128 2 ml/$152 2 mg/$139 2 ml/$190 1 ml/$207

Rat IgG (H+L) Rabbit 31218 318342 mg/$95 1.5 mg/$100

Rat IgG (H+L) Rabbit 31219 31836(min x Ms Sr Prot)* 0.5 mg/$123 0.5 mg/$128

Anti-RAT Rat IgG (H+L) Rabbit 31227F(ab′)2 Fragment 1 mg/$101of Host Antibody

Rat IgG + IgM Goat 31625(H+L) 1 mg/$143

Rat IgG (H+L) Mouse 31225 31633 31682(min x Ms Sr Prot)* 1 mg/$118 0.5 mg/$121 0.5 mg/$121

Anti-SHEEP Sheep IgG (H+L) Rabbit 31240 31840 31627 31480 313602 mg/$111 1.5 mg/$115 1.5 mg/$88 1.5 ml/$125 1 ml/$143

Sheep IgG (Fc) Rabbit 31241 31841 31441 313562 mg/$112 1.5 ml/$133 1.5 ml/$141 1 ml/$152

Sheep IgG [F(ab′)2] Rabbit 31244 31844 31481 313442 mg/$96 1.5 ml/$136 1.5 ml/$136 1 ml/$152


38



DyLight™ ConjugatesExcellent alternatives to CyDye™ Fluors.

The new DyLight™ Conjugates are an excellent alternative toCy3™ and Cy5™ Fluors, providing greater photostability andfluorescence over a broad range of pH values. The absorptionspectra of the DyLight™ 547 Fluor and the DyLight™ 647 Fluorclosely matches that of the Cy3™ and Cy5™ Fluors, respectively,enabling a seamless transition from CyDye™ to the DyLight™

Fluors without purchasing new filters.

Highlights:• Molar ratio (dye:protein) optimized to provide excellent

fluorescent intensity• Antibody conjugates are affinity-purified

DyLight™ Flours have demonstrated improved performancein several applications:• ELISA• Immunohistochemical (IHC) staining• Western blotting• Flow cytometry• Plate-based functional assays

DyLight™ Spectral CharacteristicsExcitation Emission Extinction

Dye (nm) (nm) Coefficient (min)DyLight™ 547 557 574 150,000 M-1 cm-1

DyLight™ 647 652 673 250,000 M-1 cm-1


Product # Description Pkg. Size Price31010 DyLight™ 547 Fluor, Goat Anti-Mouse IgG 1 ml $155

(H + L) Conjugated (1 mg/ml)31015 DyLight™ 647 Fluor, Goat Anti-Mouse IgG 1 ml $155

(H + L) Conjugated (1 mg/ml)31020 DyLight™ 547 Fluor, Goat Anti-Rabbit IgG 1 ml $155

(H+L) Conjugated (1 mg/ml)31025 DyLight™ 647 Fluor, Goat Anti-Rabbit IgG 1 ml $155

(H+L) Conjugated (1 mg/ml)21424 DyLight™ 547 Fluor, 1 ml $145

Streptavidin Conjugated (1 mg/ml)21824 DyLight™ 647 Fluor, 1 ml $145

Streptavidin Conjugated (1 mg/ml)

39PRIMARY & SECONDARY ANTIBODIES

Biotin Detection

Ordering InformationUnconjugated Enzyme-labeled Fluorescent-labeled

Avidin 21121, 10 mg, $67 Peroxidase: 21123, 2 mg, $108 Fluorescein: 21221, 5 mg, $10421128, 20 mg, $113 29994, 5 mg, $191 Phycoerythrin: 21021, 1 mg, $119

Alk. Phos: 21321, 100 units, $102NeutrAvidin™ Protein 31000, 10 mg, $112 Peroxidase: 31001, 2 mg, $185 Fluorescein: 31006, 5 mg, $149

Alk. Phos: 31002, 2 mg, $229Streptavidin 21122, 1 mg, $90 Peroxidase: 21126, 1 mg, $125 Fluorescein: 21224, 1 mg, $154

21125, 5 mg, $272 21124, 2 mg, $225 Rhodamine: 21724, 1 mg, $15021127, 5 mg, $389 Texas Red® Dye: 21624, 1 mg, $157

Alk. Phos: 21324, 1 mg, $152 R-Phycoerythrin: 21627, 1 ml, $13021323, 3 mg, $349 Allophycocyanin: 21629, 0.5 ml, $145

40

ANTIBODY LABELING


Chemical cross-linking reagents have become an invaluabletool in the scientific community. These reagents are used inpreparing antibody-enzyme conjugates and other labeled proteinreagents. After the protein is conjugated to an appropriateenzyme, it may then be used as a detection reagent in a varietyof assays and applications. A number of cross-linking methodshave been used to prepare enzyme conjugates. For example,an N -hydroxysuccinimide ester can be prepared from a ligandof interest, then reacted with a primary amine on the surface ofthe enzyme. While this method is necessary in some applications,such as those in which the ligand does not contain a primaryamine, it is not useful as a general-purpose method.

Antibody-Modification SitesAntibodies can be easily modified to contain labels such asbiotin, fluorescent tags or enzymes to create reagents forELISA, Western blotting, immunohistochemical staining andin vivo targeting. Pierce offers tools for a variety of antibody-modification strategies.

Understanding the functional groups available on an antibodyis the key to choosing the proper method for modification.

For example:

Primary amines (–NH2) are found on lysine residues and theN-terminus. These are abundant and distributed over theentire antibody.

Sulfhydryl groups (–SH) are found on cysteine residues andare formed by selectively reducing disulfide bonds in the hingeregion of the antibody.

Carbohydrate residues containing cis-diols can be oxidized(–CHO) to create active aldehydes. These are localized to the Fc region on antibodies and are more abundant on polyclonal antibodies.

Antigen-bindingsite Light Chains

Carbohydrate Carbohydrate

HingeRegion

CH2

CH3

CH2

CH3

CH1 CH1

VL

CL

VL

CLVH

S-SS-S

S-SS-SS-S

S-SS-S

S-S

S-SS-S

S-S

S-S

Fab (Fab')2

FC

SS

SS

SS

SS

VH

NH2

Amines onlysine residues

Sulfhydryls created whenantibody is reduced

NH2

NH2

NH2

Heavy Chains

Step 1. Preparation of Protein

For salt-free lyophilized protein:Dissolve in borate buffer.

Reconstitute fluorescent dye with DMF.

Add dye to the protein solution.Incubate for 1 hour.

Step 2. Labeling Reaction Step 3. Removal of Excess Fluorescent Dye

For proteins in buffers or salt solutions:a.) Sample volume of 100 µl or less: Exchange into borate buffer using Slide-A-Lyzer® MINI Dialysis Unit.

b.) Sample volume greater than 100 µl: Exchange into borate buffer using a D-Salt™ Dextran Column.

For sample volume of 100 µl or less:Exchange into PBS buffer using Slide-A-Lyzer® MINI Dialysis Unit.

For sample volume greater than 100 µl:Exchange into PBS buffer using a D-Salt™ Dextran Column.

EZ-Label™ Fluorescent Labeling KitsMake your own fluorescent-labeled antibody in less than two hours!

EZ-Label™ Kits are designed for labeling any size protein –small or large – even if you have only a small amount of yourprotein. Protein sample volumes ranging from 50 µl-1 ml canbe used, with protein concentration up to 10 mg/ml for eachreaction. EZ-Label™ Kits were specially developed and optimizedfor the most efficient labeling.

EZ-Label™ Kits contain everything you need to successfullylabel your antibody or protein:• Fluorescent dye provided in individual microtubes, eliminating

the need to weigh dye • Conveniently packaged dimethylformamide (DMF) to prepare

the fluorescent dye solution• Pre-made borate and phosphate buffers – just add water to

the powder and they are ready-to-use• Pre-packed, ready-to-use desalting columns for fast buffer

exchange when your protein sample volume is greaterthan 100 µl

• Slide-A-Lyzer® MINI Dialysis Units* for easy buffer exchangewhen your protein sample volume is less than or equal to 100 µl

• Amber reaction tubes – no handling in the dark required

Excitation EmissionEZ-Label™ Kit Wavelength (nm) Wavelength (nm)Fluorescein Protein Labeling Kit 491 518Rhodamine Protein Labeling Kit 544 576 Fluorescein Isothiocyanate 494 520(FITC) Protein Labeling KitThese kits contain sufficient reagents to perform five fluorescent labeling reactions,which use up to 10 mg/ml of protein for each reaction (50 µl-1 ml volume of protein).


Product # Description Pkg. Size Price53000 EZ-Label™ Fluorescein Protein Labeling Kit Kit $237

Sufficient for five coupling reactions.Includes: No-Weigh™ 6 x 1 mg

Pre-Measured Fluorescein Microtubes microtubesDimethylformamide (DMF) 1 mlBupH™ Borate Buffer Packs 5 packsBupH™ Phosphate Buffered Saline Packs 5 packsD-Salt™ Dextran Desalting Columns 5 columnsSlide-A-Lyzer® MINI Dialysis Unit Pack 5 unitsReaction Tubes 5 tubes

53002 EZ-Label™ Rhodamine Protein Labeling Kit Kit $237Sufficient for five coupling reactions.Includes: No-Weigh™ 6 x 0.5 mg

Pre-Measured Rhodamine Microtubes microtubesDimethylformamide (DMF) 1 mlBupH™ Borate Buffer Packs 5 packsBupH™ Phosphate Buffered Saline Packs 5 packsD-Salt™ Dextran Desalting Columns 5 columnsSlide-A-Lyzer® MINI Dialysis Unit Pack 5 unitsReaction Tubes 5 tubes

53004 EZ-Label™ Fluorescein Isothiocyanate Kit $237(FITC) Protein Labeling KitSufficient for five coupling reactions.Includes: No-Weigh™ 6 x 1 mg

Pre-Measured FITC Microtubes microtubesDimethylformamide (DMF) 1 mlBupH™ Borate Buffer Packs 5 packsBupH™ Phosphate Buffered Saline Packs 5 packsD-Salt™ Dextran Desalting Columns 5 columnsSlide-A-Lyzer® MINI Dialysis Unit Pack 5 unitsReaction Tubes 5 tubes

* Slide-A-Lyzer® MINI Dialysis Unit Technology is protected by U.S. patent # 6,039,871.

The fluorescent-labeling procedure is easy when you use EZ-Label™ Kits.

41ANTIBODY LABELING

Fluorescent Labeling

800-874-3723 • 815-968-0747 • www.piercenet.com42

ANTIBODY LABELINGEnzyme Labeling

Maleimide ActivationThe heterobifunctional cross-linker SMCC (Product # 22360)and its water-soluble analog Sulfo-SMCC (Product # 22322)have good general utility in preparing immunologically activehorseradish peroxidase or alkaline phosphatase conjugates.They are most useful when preparing conjugates of reducedIgG and F(ab′)2, because these methods involve the initial stepof preparing a maleimide-activated (sulfhydryl-reactive) enzymederivative. Studies have shown that the two-step maleimidemethod is superior to glutaraldehyde or meta-periodate methodsfor enzyme conjugation (Figure 15). The maleimide method giveshigher yields with less polymerization, producing a conjugatepreparation with superior immunoassay characteristics.

Maleimide-activated enzymes can be prepared using theheterobifunctional cross-linker Sulfo-SMCC. This reagent containsan N-hydroxy-sulfosuccinimide (Sulfo-NHS) functional groupand a maleimide functional group and it is water-soluble dueto the presence of the sulfonate (–SO3

-) group on the N-hydrox-ysuccinimide ring. The sulfonate group also contributes to thestability of the molecule in aqueous solution. A study of thehydrolysis rate of the maleimide functional group from Sulfo-SMCCshowed that it is less prone to hydrolysis to the maleamic acidthan the non-sulfonated SMCC. The maleimide groups ofSulfo-SMCC exhibit no decomposition at pH 7 at 30˚C within6 hours. The Sulfo-NHS ester group reacts with primary amineson the enzyme surface to form a stable amide bond. After thisfirst step of conjugation, the enzyme will have maleimide groupson its surface that react optimally toward sulfhydryl groupsbetween pH 6.5 and 7.5 to form stable thioether bonds. Maleimide-mediated conjugation strategies are summarized in Figure 15.

SHProtein

Protein Protein

Three methods for free sulfhydryl generation Maleimide activation of enzyme

Native protein has a freesulfhydryl on its surface.

Native protein is reacted with SATA. Blocked sulfhydryl groups are introduced on primary amines.Hydroxylamine (H) treatment generates free sulfhydryls.

Native protein contains disulfide bonds that can be reduced to generate free sulfhydryls. Enzyme is SMCC-labeled through primary amines to generate amaleimide-activated enzyme for conjugation to free sulfhydryls.

O

C

S S

MEAEDTA

SH

NH2Protein SATA

Protein

Protein

CH2 S

O

C CH3H

CH2 SH

SHProtein

Protein

H

NO

C

H

N

O

O

OO

OO

N

SMCC

NHS

OH

O

O

N

N+ H2N

S

O

ON

O N E

E

O

ON

O N

SH

E

1

2

3

Figure 15. Three strategies for maleimide-mediated conjugation of enzymes.

ANTIBODY LABELING 43

Two reagents, Mercaptoethylamine•HCl (Product # 20408)and SATA (Product # 26102), are available to produce freesulfhydryls on macromolecules for conjugation to themaleimide-activated enzymes. For labeling antibody molecules,mild reduction with Mercaptoethylamine•HCl (MEA) results intwo half-antibody fragments containing free sulfhydryl groupsin the hinge region. Labeling in this area is advantageous becauseit directs the modification away from the antigen-binding region.Native proteins lacking a free sulfhydryl on their surface canbe reacted with SATA to generate blocked sulfhydryl groups.The SATA molecule reacts with primary amines via its NHS esterend to form stable amide linkages. The acetylated sulfhydrylgroup (blocked) is stable until treated with hydroxylamine togenerate the free sulfhydryls.

Pierce offers stable, preactivated enzyme derivatives that arereactive toward sulfhydryl (–SH) groups, EZ-Link™ MaleimideActivated Alkaline Phosphatase (Product # 31486) and HorseradishPeroxidase (Product # 31485). These products eliminate thefirst step of the two-step maleimide method, simplifying andfacilitating the conjugation protocol, while saving several hours.They can be used to prepare enzyme conjugates directly fromproteins, peptides or other ligands that contain a free –SH group.Two reagents, SATA and mercaptoethylamine•HCl, are alsoincluded in the kit formats to produce free sulfhydryls onmacromolecules for conjugation.

EZ-Link® Maleimide Activated Peroxidase ReferencesChoi, J.Y., et al. (2002). J. Biol. Chem. 277, 21630-21638. Seo, Y.R., et al. (2002). Proc. Natl. Acad. Sci. 99, 14548-14553. Yoo, J.H., et al. (2004). J. Biol. Chem. 279, 848-858.


Product # Description Pkg. Size Price31486 EZ-Link® Maleimide Activated 2 mg $174

Alkaline Phosphatase31493 EZ-Link® Maleimide Activated Kit $359

Alkaline Phosphatase KitIncludes: EZ-Link® Maleimide 2 mg

Activated Alkaline PhosphataseActivation/Conjugation Buffer 20 mlBupH™ Tris Buffered Saline Pack 2 packsBupH™ Phosphate Buffered Saline Pack 1 packPolyacrylamide Desalting Column 1 x 10 mlMercaptoethylamine•HCl 6 mgSATA 2 mgHydroxylamine 5 mgDMF 1 mlColumn Extender

31485 EZ-Link® Maleimide Activated 5 mg $132Horseradish Peroxidase

31494 EZ-Link® Maleimide Activated Kit $317Horseradish Peroxidase KitIncludes: EZ-Link® Maleimide 5 mg

Activated Horseradish PeroxidaseActivated Horseradish Peroxidase 20 mlConjugation Buffer

2-Mercaptoethylamine•HCl 6 mgSATA 2 mgDimethylformamide 1 mlHydroxylamine•HCl 5 mgPolyacrylamide Desalting Column 1 x 10 ml

23460 Protein-Coupling Handle Addition Kit Kit $181Includes: SATA 2 mg

Hydroxylamine•HCl 5 mgConjugation Buffer Stock (10X) 20 mlBupH™ Pack PBS 1 packDimethylformamide (DMF) 1 mlD-Salt™ Dextran Desalting Column 1 x 5 mlColumn Extender 1Ellman’s Reagent (DTNB) 2 mgCysteine•HCl H2O 20 mg

44

ANTIBODY LABELING

Figure 16. Conjugation scheme for periodate oxidation and subsequentreductive amination.

PeriodateGlycoproteins such as horseradish peroxidase and glucoseoxidase and most antibody molecules can be activated forconjugation by treatment with periodate. Oxidizing polysac-charide residues in a glycoprotein with sodium periodate providesa mild and efficient way of generating reactive aldehyde groupsfor subsequent conjugation with amine- or hydrazide-containingmolecules via reductive amination (Figure 16). Some selectivityof monosaccharide oxidation may be accomplished by regulatingthe concentration of periodate in the reaction medium. In thepresence of 1 mM sodium periodate, sialic acid groups arespecifically oxidized at adjacent hydroxyl residues, cleaving offtwo molecules of formaldehyde and leaving one aldehyde group.At higher concentrations of sodium periodate (10 mM or greater),other sugar residues will be oxidized at points where adjacentcarbon atoms contain hydroxyl groups. This reaction shouldbe performed in the dark to prevent periodate breakdown andfor a limited period of time (15-30 minutes) to avoid loss ofenzymatic activity.

Cross-linking with an amine-containing protein takes placeunder alkaline pH conditions through the formation of Schiffbase intermediates. These relatively labile intermediates canbe stabilized by reduction to a secondary amine linkage withsodium cyanoborohydride. Reductive amination has been done

using sodium borohydride or sodium cyanoborohydride; however,cyanoborohydride is the better choice because it is more specificfor reducing Schiff bases and will not reduce aldehydes. Smallblocking agents such as lysine, glycine, ethanolamine or Tris canbe added after conjugation to quench any unreacted aldehydesites. Ethanolamine and Tris are the best choices for blockingagents because they contain hydrophilic hydroxyl groups withno charged functional groups.

The pH of the reductive amination reaction can be controlledto affect the efficiency of the cross-linking process and thesize of the resultant antibody-enzyme complexes formed. Atphysiological pH, the initial Schiff base formation is less efficientand conjugates of lower molecular weight result. At more alkalinepH (i.e., pH 9-10), Schiff base formation occurs rapidly and withhigh efficiency, resulting in conjugates of higher molecularweight and greater incorporation of enzyme when oxidizedenzyme is reacted in excess. Low molecular weight conjugatesmay be more optimal for immunohistochemical staining orblotting techniques in which penetration of the complex throughmembrane barriers is an important consideration. Washing stepsalso more effectively remove excess reagent if the conjugate isof low molecular weight, thus maintaining low background inan assay. By contrast, conjugates of high molecular weight aremore appropriate for ELISA procedures in a microplate format,where high sensitivity is important and washing off excessconjugate is not a problem.

GlutaraldehydeAnother method for conjugation uses glutaraldehyde, one of theoldest homobifunctional cross-linking reagents used for proteinconjugation. It reacts with amine groups to create cross-links byone of several routes. Under reducing conditions, the aldehydeson both ends of glutaraldehyde will couple with amines to formsecondary amine linkages. The reagent is highly efficient at proteinconjugation but has a tendency to form various high-molecularweight polymers, making results difficult to reproduce.

EZ-Link® Activated Peroxidase ReferencesSandt, C.H. and Hill, C.W. (2001). Infect. Immun. 69, 7293-7303. Turpin, E.A., et al. (2003). J. Clin. Microbiol. 41, 3579-3583.

EZ-Link® Plus Activated Peroxidase ReferencesGlover, L. (2002). Eur. J. Biochem. 269, 4607-4616. Nawa, M., et al. (2000). Clin. Diagn. Lab. Immunol. 7, 774-777. Völkel, T., et al. (2001). Protein Eng. 14, 815-823.

OHHO

RO

HOO

O

OO

RO

HOO

O

NNH2

NaCNBH3

O

ROHO

O

O

Enzyme containingpolysaccharide chains

Enzyme withreactive aldehyde groups

Reductive amination formsstable secondary amine linkage

Antibody moleculecontaining amine groups

NalO4

(oxidation)E E

E


45ANTIBODY LABELING

Alkaline PhosphataseA highly sensitive enzyme for ELISA and immunohistochemical applications.

Highlights:• Purified form – ready to conjugate without prior dialysis• Activity is not affected by exposure to antibacterial agents

such as sodium azide or thimerosal• Specific activity > 2,000 units/mg• One unit is defined as the amount that will hydrolyze

1.0 µmole of p -nitrophenyl phosphate per minute at 37°C in 1.0 M diethanolamine, 0.5 mM MgCl2, pH 7.8

Specific Activity per mg ProteinBuffer 25°C 37°C0.1 M Glycine, 1.0 mM ZnCl2, 1.0 mM MgCl2, > 500 > 1,0006.0 mM p -Nitrophenyl phosphate, pH 10.41.0 M Diethanolamine, 0.5 mM MgCl2, > 1,000 > 2,00015 mM p -Nitrophenyl phosphate, pH 9.8

ReferencesBulman, A.S. and Heyderman, E. (1981). J. Clin. Pathol. 34, 1349-1351.Cordell, J.L., et al. (1984). J. Histochem. Cytochem. 32, 219-229.Yolken, R.H. (1982). Rev. Infect. Dis. 4, 35-68.


Product # Description Pkg. Size Price31391 ImmunoPure® Alkaline Phosphatase 20 mg $ 652

Calf intestinal. Supplied in Tris Buffer, pH ~7Triethanolamine, 1 mM MgCl2, 3 M NaCl, pH 7.6

31392 ImmunoPure® Alkaline Phosphatase 100 mg $2,528

Horseradish PeroxidaseHigh-specific enzyme activity makes it the enzyme of choice.

Highlights:• Superior to alkaline phosphatase and β-galactosidase conjugates

due to the higher specific enzyme activity• Small size (40 kDa) allows excellent cellular penetration• Variety of substrates available• Ideal in blotting and cytochemistry applications• Used as the reporter enzyme for SuperSignal®

Chemiluminescent Substrates

ReferencesCordell, J.L., et al. (1984). J. Histochem. Cytochem. 32, 219-229.Hosoda, H., et al. (1987). Chem. Pharm. Bull. 35, 3336-3342.Passey, R.B., et al. (1977). Clin. Chem. 23(1), 131-139.Porstmann, B., et al. (1985). J. Immunol. Methods. 79, 27-37.Samoszuk, M.K., et al. (1989). Antibody, Immunoconjugates andRadiopharmaceuticals 2, 37-46.Wordinger, R.J., et al. (1987). Manual of Immunoperoxidase Techniques, 2nd Edition.Chicago: American Society of Clinical Pathologists Press, pp. 23-24.Yolken, R.H. (1982). Rev. Infect. Dis. 4(1), 35-68.


Product # Description Pkg. Size Price31490 ImmunoPure® Horseradish Peroxidase 10 mg $ 5331491 ImmunoPure® Horseradish Peroxidase 100 mg $190

U.S.Product # Description Pkg. Size Price31487 EZ-Link® Plus Activated Peroxidase 1 mg $ 63

(Periodate Activated)31488 EZ-Link® Plus Activated Peroxidase 5 x 1 mg $232

(Periodate Activated)31489 EZ-Link® Plus Activated Peroxidase Kit Kit $317

(Periodate Activated)Includes: EZ-Link® Plus Activated Peroxidase 5 x 1 mg

Sodium Cyanoborohydride Solution 1 x 0.5 mlQuenching Buffer 25 mlBupH™ Phosphate Buffered Saline Pack 500 mlBupH™ Carbonate Buffer Pack 500 ml

U.S.Product # Description Pkg. Size Price31496 EZ-Link® Activated Peroxidase 1 mg $ 58

(Glutaraldehyde Activated)31495 EZ-Link® Activated Peroxidase 5 mg $160

(Glutaraldehyde Activated)31497 EZ-Link® Activated Peroxidase Kit $317

Antibody Labeling Kit(Glutaraldehyde Activated)Includes: EZ-Link® Activated Peroxidase 5 mg

Conjugation Buffer 50 mlLysine 250 mgImmobilized Protein A/G Column 0.5 mlGentle Ag/Ab Binding Buffer 200 mlGentle Ag/Ab Elution Buffer 200 ml

Ordering Information

Pierce offers an extensive line of substrates for AP and HRP,depending upon the plate-reading equipment available and thelevel of sensitivity required in the ELISA. Chemiluminescentand chemifluorescent substrates provide a stronger signalthan colorimetric substrates, thus providing greater sensitivity.

ELISA conditions must always be re-optimized when switchingto a more sensitive substrate (Table 7). Greater dilutions of anHRP-conjugate should be used with a chemiluminescentsubstrate than with a colorimetric substrate.

46

CHOOSING A SUBSTRATE


Table 7. Properties of ELISA substrates for horseradish peroxidase (HRP) and alkaline phosphatase (AP).

Dilution range of Ab ApproximateSubstrate Product # Page Measurement - Color (From 1 mg/ml stock) Sensitivity* EnzymeSuperSignal® ELISA Femto 37075 50 425 nm chemiluminescent 1° 1:10K – 1:20K 0.17 pg/well HRP

2° 1:50K – 1:100KSuperSignal® ELISA Pico 37070 49 425 nm chemiluminescent 1° 1:1K 0.5 pg/well HRP

2° 1:10KQuantaBlu™ Substrate 15169 51 325 nm/420 nm chemifluorescent 1° 1:10K – 1:20K 0.5 pg/well HRP

2° 1:50K – 1:100K1-Step™ Ultra TMB 34028 53 450 nm stopped – Yellow 1° 1:500 – 1:1K 2 pg/well HRP

652 nm nonstop – Blue 2° 1:4K – 1:100K1-Step™ Turbo TMB 34022 53 450 nm stopped – Yellow 1° 1:500 – 1:1K 7 pg/well HRP

652 nm nonstop – Blue 2° 1:4K – 1:100K1-Step™ Slow TMB 34024 53 450 nm stopped – Yellow 1° 1:500 – 1:1K 8 pg/well HRP

652 nm nonstop – Blue 2° 1:4K – 1:100KTMB Substrate Kit 34021 53 450 nm stopped – Yellow 1° 1:500 – 1:1K 5.5 pg/well HRP

652 nm nonstop – Blue 2° 1:4K – 1:100K1-Step™ ABTS 37615 52 410 nm/650 nm – Green 1° 1:500 – 1:1K 0.25 ng/well HRP

2° 1:4K – 1:50KABTS 34026 52 410 nm/650 nm – Green 1° 1:500 – 1:1K 0.25 ng/well HRP

2° 1:4K – 1:50KOPD Powder 34005 52 490 nm stopped – Green 1° 1:500 – 1:1K 7 pg/well HRP

450 nm nonstop – Yellow-Orange 2° 1:4K – 1:100KOPD Tablets 34006 52 490 nm stopped – Green 1° 1:500 – 1:1K 7 pg/well HRP

450 nm nonstop – Yellow-Orange 2° 1:4K – 1:100K1-Step™ PNPP 37621 54 405 nm – Yellow 1° 1:500 10 ng/well AP

2° 1:5KPNPP Kit 37620 54 405 nm – Yellow 1° 1:500 10 ng/well AP

2° 1:5KPNPP Tablets 34047 54 405 nm – Yellow 1° 1:500 10 ng/well AP

2° 1:5KPNPP Powder 34045 54 405 nm – Yellow 1° 1:500 10 ng/well AP

2° 1:5K* Actual sensitivity is unique to each antibody-antigen pair. The approximate sensitivities listed are conservative amounts that should be easily detectable for most antigens.

Horseradish Peroxidase Substrates

Horseradish peroxidase is a 40 kDa protein that catalyzesthe oxidation of substrates by hydrogen peroxide, resulting ina colored or fluorescent product or the release of light as abyproduct. HRP functions optimally at a near-neutral pH andcan be inhibited by cyanides, sulfides and azides. Antibody-HRPconjugates are superior to antibody-AP conjugates with respectto the specific activities of both the enzyme and antibody. Inaddition, a high turnover rate, stability, low cost and wideavailability of substrates make HRP the enzyme of choicefor most applications.

When selecting a substrate for HRP-based ELISAs, there are anumber of possibilities. The question of sensitivity is often theoverriding factor in making a selection. However, considerationshould also be given to whether the substrate contains harmfulsolvents, what detection equipment is available and how theassay will be measured (kinetic or end-point). In many ELISAapplications, colorimetric substrates provide a sufficient levelof sensitivity and dynamic range. This is evident with the PierceEndogen Cytokine ELISA Kits, in which a TMB substrate coupledwith a streptavidin-HRP detection system results in pg/mlsensitivity. Detection below this level requires a fluorescent orchemiluminescent signal. Characteristics of the HRP ELISAsubstrates offered by Pierce are summarized in Table 7.

Chromogenic ELISA substrates result in a soluble, coloredproduct. These substrates are used in most ELISAs becausedetection of a colored product can be performed on a spec-trophotometric plate reader. TMB (3,3′,5,5′-tetramethylbenzidine)is the most common chromogenic substrate for HRP and isavailable in several formats. 1-Step™ Ultra TMB (Product #34028) yields the greatest sensitivity among the TMB substrates,followed by 1-Step™ Turbo TMB (Product # 34022) and 1-Step™

Slow TMB (Product # 34024). The 1-Step™ Substrates arepreformulated so no mixing or pre-filtering is required. Althoughthe sensitivity is lower, the 1-Step™ Slow TMB and 1-Step™ ABTS(2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammoniumsalt) are ideal for kinetic readings. OPD (o -phenylenediaminedihydrochloride, Product # 34005) is another relatively sensitiveHRP substrate that produces a yellow-orange color.

The greatest sensitivity in ELISA applications is obtainedusing chemiluminescent or chemifluorescent substrates. Thesesubstrates have been steadily gaining in popularity becauseof their sensitivity (less than 1 pg/ml), large linear range fordetection and excellent antibody conservation. Pierce offersthe chemiluminescent SuperSignal® ELISA Pico (Product #37070) and SuperSignal® ELISA Femto (Product # 37075)Substrates and the chemifluorescent QuantaBlu™ Substrate(Product # 15169).

Enzyme-labeled reagents are detected using chromogenic,chemiluminescent or chemifluorescent substrates. Chromogenicsubstrates are generally the least expensive, while luminescentor fluorescent substrates are often more sensitive. Whenperforming ELISAs, a soluble substrate is used and converted

to a soluble end product. ELISAs are performed on polystyreneplates, and the enzyme levels are determined by monitoringsignal development with a spectrophotometer (a luminometerfor luminescence or a fluorometer for fluorescence).

Choosing a Detection Signal TypeColorimetric Substrates Chemifluorescent Substrates Chemiluminescent Substrates

ELISA • Medium/low sensitivity • High sensitivity • High sensitivity• Generally less expensive • Generally more expensive • Generally more expensive• Many substrates available • Few substrates available • Few substrates available• Slow signal generation • Rapid signal generation • Rapid signal generation• Enzyme catalyzed quickly • Enzyme activity maintained • Enzyme catalyzed quickly• Small linear range/poor low-end linearity • Large linear range/enhanced low-end linearity • Large linear range/enhanced low-end linearity• Flexible (stopped, nonstopped and • Flexible (stopped, nonstopped and • Nonflexiblekinetic assays) kinetic assays)

Detection Spectrophotometer provides Fluorometer provides Luminometer provides Equipment quantifiable results quantifiable results quantifiable results

47CHOOSING A SUBSTRATE

Figure 17. Comparison of QuantaBlu™ Substrate to other substrates.QuantaBlu™ Substrate and the colorimetric substrates were incubated for30 minutes at RT, followed by addition of a stop solution. QuantaBlu™

Substrate gave the greatest signal:noise (S/N) ratios and exhibited thelowest detection limit.

When energy in the form of light is released from a substancebecause of a chemical reaction, the process is called chemilumines-cence. Luminol is one of the most widely used chemiluminescentreagents and its oxidation by peroxide results in creation of anexcited state product called 3-aminophthalate. This product decaysto a lower energy state by releasing photons of light (Figure 18).

Figure 18. Luminol is oxidized in the presence of horseradish peroxidase andhydrogen peroxide to form an excited state product (3-aminophthalate).The 3-aminophthalate emits light at 425 nm as it decays to the ground state.

Chemiluminescent substrates have steadily gained in popularitythroughout the past decade because they offer several advantagesover other detection methods. These advantages have allowedchemiluminescence to become the detection method of choicein most protein laboratories. Using chemiluminescence allowsmultiple exposures to be performed to obtain the best image.A large linear response range allows detection and quantitation

over a large range of protein concentrations. Most importantly,chemiluminescence yields the greatest sensitivity of any availabledetection method. Using HRP as the enzyme label and SuperSignal®

ELISA Femto Chemiluminescent Substrate (Product # 37075),lower detection limits in the upper femtogram range are possiblebecause the enhancers in this substrate greatly intensify theemitted light and extend the signal duration.

Chemiluminescent substrates differ from other substrates inthat the light detected is a transient product of the reaction thatis only present while the enzyme-substrate reaction is occurring.This is in contrast to substrates that produce a stable, coloredproduct; these colors persist in the well after the enzyme-substratereaction has terminated. In a chemiluminescent ELISA, thesubstrate is the limiting reagent in the reaction; as it is exhausted,light production decreases and eventually ceases. A well-optimized procedure using the proper antibody dilutions willproduce a stable output of light, allowing consistent andsensitive detection of proteins. When the antibody is not dilutedsufficiently, too much enzyme is present and the substrate isused up quickly. A stable output of light will never be achieved.This is the single greatest cause of variability in chemilumi-nescent ELISAs. To avoid this problem, it is crucial to optimizethe amount of antibody used for detection. Antibody supplierstypically suggest a dilution range for using their antibody in anELISA. This dilution range is often appropriate for experimentsdetected with a relatively insensitive chromogenic substrate,but a much greater dilution is generally required for optimumperformance with a sensitive chemiluminescent substrate.

Table 8. Advantages of enhanced chemiluminescence.

Sensitive • Intense signal with low background• Requires less antigen and antibody

Fast • Rapid substrate processing• Signal generated within seconds

Nonhazardous • No health hazards• No waste disposal problems

Stable • Unlike radioisotopes, the shelf life is long• Store at 4°C

Large linear • Can detect a large range of protein concentrationsresponseQuantitative • Results can be scanned using an imaging device, such

as a CCD camera

H2O2 + +HRPLIGHT

*

@ 425 nm

NH

NH

O

ONH2

O

O

O

ONH2

O

O

O

ONH2

S/N

Ratio

Biotinylated-HRP, pg/well

ABTSOPDTMB

QuantaBlu™ Substrate

0 1,000 2,000 3,000 4,000

90

80

70

60

50

40

30

20

10

0

48



SuperSignal® ELISA Pico Chemiluminescent SubstrateExperience the same sensitivity in your luminometer that you’ve come to expect from all Pierce SuperSignal® Products.

SuperSignal® ELISA Pico Chemiluminescent Substrate is optimizedfor luminometer-based assays to generate an intense light signal.

Figure 19. Immediate light generation with SuperSignal® ELISA PicoChemiluminescent Substrate. 200 pg of biotinylated HRP or biotinylated APwere added to separate wells of Reacti-Bind™ NeutrAvidin™ Coated WhitePolystyrene Plates. The plates were then incubated for 30 minutes at roomtemperature (RT) on a plate shaker and then each well was washed threetimes with BupH™ Tris Buffered Saline. Working solutions of chemiluminescentsubstrates were prepared according to the manufacturers’ instructions. ForSuperSignal® ELISA Pico Chemiluminescent Substrate and anotherluminol-based system (Brand A), 100 µl of each substrate working solutionwas added to the appropriate plate well. For the dioxetane-based system,wells were washed with 1X Assay Buffer. Next, 100 µl of the dioxetaneworking solution was added to the appropriate plate well. All plates wereincubated on a plate shaker at RT for 1 minute. Plates were then read on aplate luminometer with a 0.2 second read time per well. Several readingswere taken over a 30-minute period.

Highlights:• Immediate light generation – intense signal is produced

immediately at room temperature or at 37°C• High signal:noise ratio – minimal background• Low picogram sensitivity – detect proteins in your ELISAs

down to the picogram levels• Room temperature storage – a consistent product with

ambient shipping and no need to store at 4°C• 8-hour working solution stability – consistent performance

of the working solution over an 8-hour period with only a10% decrease in activity at 24 hours

• Flexible – signal can be read in black or white opaque plates• Emits light at 425 nm

ReferencesBradley, K.A., et al. (2004). J. Biol. Chem. 278, 49342-49347. McKevitt, M., et al. (2003). Genome Res. 13, 1665-1674.


Product # Description Pkg. Size Price37070 SuperSignal® ELISA Pico 100 ml $195

Chemiluminescent Substrate*Includes: Luminol/Enhancer 50 ml

Stable Peroxide Buffer 50 ml*SuperSignal® Technology is protected by U.S. patent # 6,432,662.

RLU

Time (minutes)

Dioxetane-Based, Brand BLuminol-Based, Brand ASuperSignal® ELISA Pico

Chemiluminescent Substrate

0 10 20 30

700

600

500

400

300

200

100

0

Kinetic Analysis of 200 pg of Biotinylated HRP

HRP Substrates for ELISAExcellent sensitivity for use in luminometers.

Researchers looking for greater sensitivity in their ELISAs orany other solution-based assay are now able to use chemilu-minescent substrates for enzyme detection and quantification.These ELISAs can take place in either a test tube or a microplateand are quantified by measuring relative light units (RLU) in aluminometer. SuperSignal® ELISA Pico Chemiluminescent

Substrate was developed for researchers who need highsensitivity at an economical price. SuperSignal® ELISA FemtoMaximum Sensitivity Chemiluminescent Substrate uses animproved enhancer system that meets the needs of high-throughput screening and diagnostic customers. Both havetheir own unique features as listed in the table.

Table 9. Which substrate is right for you?

Working Solution StabilitySubstrate Detection Limits Kinetics at Room TemperatureSuperSignal® ELISA Femto Maximum • Quantitative to the femtogram level • Immediate light generation • 6-hour working solution stabilitySensitivity Substrate • 5- to 30-minute stability,

depending on HRP concentrationSuperSignal® ELISA Pico • Quantitative to the picogram level • Immediate light generation • 8-hour working solution stabilityChemiluminescent Substrate • 30-minute stable light output • Only 10% loss of activity after 24 hours


50


SuperSignal® ELISA FemtoMaximum SensitivitySubstrate utilizes theSuperSignal® Systemwith a new enhancerformulation for superiorprotein detection and low-end linearity in ELISAapplications.

SuperSignal® ELISA Femto Substrate’s rapid signal generationhas the added benefit of decreasing the substrate incubationperiod. SuperSignal® ELISA Femto Substrate generates detectablelight within 1 minute of addition to trace amounts of solubleHRP, saving up to 30 minutes per assay. This feature makesSuperSignal® ELISA Femto Substrate ideal for high-throughputscreening (HTS) applications in which as many as 100,000assays may be run daily on robotic equipment. As incubationperiods are often a limiting factor in the development of rapidautomated assays, SuperSignal® ELISA Femto Substrate is thelogical choice for automated HTS applications.

Highlights:• Immediate light generation – intense signal generated

immediately at both room temperature and 37°C• Improved low-end linearity – easy detection of low quantities

of proteins with high signal:noise ratios and low-end linearityof dose response curves

• High sensitivity – femtogram-level detection of targetproteins in an ELISA

• Reduction in assay time – high sensitivity allows for reductionin ELISA incubation steps

• Stability – storage for six months at room temperature or aminimum of 12 months at 4°C with a six-hour workingsolution stability

• Emits light at 425 nm

Incubation Time Required to Reach Maximum SignalSuperSignal® ELISA Femto 1 minute at room temperatureMaximum Sensitivity SubstrateSuperSignal® ELISA Pico 1 minute at room temperatureChemiluminescent SubstrateDioxetane-based Substrate System 30 minutes at room temperatureLuminol-based Substrate System 1 minute at room temperatureAcridan-based Substrate System 5 minutes at room temperatureTMB Colorimetric Substrate 15 minutes at room temperature

Figure 20. Femtogram detection of target protein and superior low-endlinearity. The dose response curve generated from an IL-2 ELISA illustratesthe exceptional low-end linearity achieved with SuperSignal® ELISA FemtoSubstrate and the incredible sensitivity attainable. The SuperSignal® ELISAFemto Substrate detected down to 168 fg of IL-2. The R2 value of the curvewas calculated to be 1.00 for signal generated at less than 1,600 fg of IL-2.

Figure 21. Signal intensity and kinetics comparison of chemiluminescentsubstrates. The dose response curve using SuperSignal® ELISA FemtoMaximum Sensitivity Substrate in an IL-2 ELISA was compared to curvesgenerated with a dioxetane-based substrate, another luminol-basedsubstrate, an acridan-based system and TMB. SuperSignal® ELISA FemtoMaximum Sensitivity Substrate demonstrated immediate light generationwith maximum peak intensity and high RLU values.

ReferencesBrandt, E.B., et al. (2003). J. Clin. Invest. 112, 1666-1677. Hanley, N.R.S. and Hensler, J.G. (2002). J. Pharmacol. Exp. Ther. 300, 468-477.Masri, H.P. and Cornellissen, C.N. (2002). Infect. Immun. 70, 732-740.Su, S.V., et al. (2004). J. Biol. Chem. In press.


Product # Description Pkg. Size Price37075 SuperSignal® ELISA Femto 100 ml $205

Maximum Sensitivity Substrate*Includes: Luminol/Enhancer 50 ml

Stable Peroxide Solution 50 ml*SuperSignal® Technology is protected by U.S. patent # 6,432,662.

Net R

LU

pg IL-20 5 10 15 2520

1,500

1,200

900

600

300

0

SuperSignal® ELISA Femto Substrate SuperSignal® ELISA Pico Substrate

Dioxetane-based SubstrateLuminol-based SubstrateAcridan-based Substrate

Net R

LU

fg IL-20 500 1,000 1,500

120

100

80

60

40

20

0

SuperSignal® ELISA Femto Maximum Sensitivity SubstrateThe most powerful substrate for high-throughput screening/diagnostic applications with high sensitivity and superior low-end linearity.

51

QuantaBlu™ Fluorogenic Peroxidase SubstratesThe ideal fluorescent substrates for use with peroxidase enzymes.

A variety of substrates areavailable for detectingperoxidase activity inELISA-based assays.Colorimetric substrates(e.g., TMB, OPD andABTS) have been usedwidely for years. Each ofthese substrates variesgreatly with respect to its

performance characteristics such as detection sensitivity,working range and attainable signal:noise ratios. Substrateflexibility is also a key issue that affects an assay. Stability,development time requirements and the capacity to performstopped and/or kinetic assays vary significantly among thesesubstrates. Ideally, a substrate is stable, is very sensitive, hashigh attainable signal:noise ratios, possesses a broad dynamicrange, and allows the user to perform stopped, nonstoppedand kinetic assays. QuantaBlu™ Fluorogenic Peroxidase Substratemeets all the requirements of an ideal substrate for use inperoxidase detection.

QuantaBlu™ Substrate generates a blue fluorescent productupon reaction with peroxidase. The fluorescent product doesnot photobleach. Fluorometric-based detection overcomes thelimitations of colorimetric substrate detection, which does notallow for quantitation of greater than four optical density units.QuantaBlu™ Substrate allows for stopped, nonstopped and kineticassays to be performed. Incubation times with QuantaBlu™

Substrate for stopped and nonstopped assays may be variedbetween 1 to 90 minutes at either room temperature or 37°C.QuantaBlu™ Substrate exhibits a flat baseline in assays, whichfacilitates low-level detection sensitivity and allows for highsignal:noise ratios.

QuantaBlu™ Fluorogenic Peroxidase Substrate provides the abilityto rapidly detect peroxidase at very low concentrations in shortperiods of time. Figure 23 illustrates the detection of peroxidasefrom 0-10 pg per well from 1.5 to 6.5 minutes of substrateincubation time. At cycle 1 (1.5 minute incubation) as little as2.5 pg of peroxidase could be detected, while at cycle 6 (6.5minute incubation) 0.625 pg of peroxidase could be detected.

Highlights:• More sensitive than TMB, OPD or ABTS substrates• Flexible stopped, nonstopped or kinetic assays possible• Large dynamic range (4 log peroxidase concentration range)• Excellent stability – working solution is stable for 24 hours• Large Stokes’ shift; excitation/emission maxima of 325/420;

range of 315-345/370-460• Does not photobleach

Figure 22. Comparison of QuantaBlu™ Substrate to other substrates.QuantaBlu™ Substrate and the colorimetric substrates were incubated for 30minutes at RT, followed by addition of a stop solution. QuantaBlu™

Substrate gave the greatest signal:noise (S/N) ratios and exhibited thelowest detection limit.

Figure 23. Rapid and sensitive detection with QuantaBlu™ FluorogenicPeroxidase Substrate. Detection of bound biotinylated peroxidase at 0-10pg/well using QuantaBlu™ Substrate. QuantaBlu™ Fluorogenic PeroxidaseSubstrate was read in a nonstopped mode using a 1-minute instrumentcycle time between reads.

ReferencesAtamna, H., et al. (2000). Proc. Natl. Acad. Sci. 97, 686-691. Ayala, P., et al. (2002). Infect. Immun. 70, 5965-5971. Jefcoat, A.M., et al. (2001). Am J Physiol Lung Cell Mol Physiol. 281, L704-712. Savage, M.D., et al. (1998). Previews 2(1), 6-9.Savage, M.D., et al. (1998). Previews 2(2), 18-19.


Product # Description Pkg. Size Price15169 QuantaBlu™ Fluorogenic Kit $169

Peroxidase Substrate*Includes: QuantaBlu™ Substrate 250 ml

QuantaBlu™ Stable Peroxide Solution 30 mlQuantaBlu™ Stop Solution 275 ml

15162 QuantaBlu™ NS/K Substrate Kit $140(for nonstopped and kinetic assays)Includes: QuantaBlu™ Substrate 250 ml

QuantaBlu™ Stable Peroxide Solution 30 ml* QuantaBlu™ Fluorogenic Peroxidase Assay Technology is protected by

U.S. patent # 6,040,150.

S/N

Ratio


4 minutes5 minutes6 minutes

1 minutes2 minutes3 minutes

0 2 4 6 8 10

4.0

3.5

3.0

2.5

2.0

1.5

1.0

S/N

Ratio


ABTSOPDTMB

QuantaBlu™ Substrate

0 1,000 2,000 3,000 4,000

90

80

70

60

50

40

30

20

10

0

OPD

OPD yields a water-soluble yellow-orange product when reactedwith peroxidase with an absorbance maximum of 492. OPDcan easily be dissolved in a substrate buffer such as StablePeroxide Substrate Buffer (Product # 34062).


Product # Description Pkg. Size Price34005 OPD 25 g powder $ 4534006 OPD Tablets 50 tablets (5 mg/tablet) $104

ABTS

ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is a water-soluble HRP substrate that yieldsa green end product upon reaction with peroxidase. The greenproduct has two major absorbance peaks, 405 nm and 650 nm.ABTS is less sensitive than OPD and TMB in ELISA applications.It is less readily oxidized, and its color development is slower(approximately 20 minutes). This may be advantageous ifunacceptable background results from the use of the OPD orTMB substrates due to higher sensitivities.

Highlights:• One component• Ready-to-use• Excellent choice when maximum sensitivities are not required• Slow reaction that can easily be followed with a kinetic reader

ReferencesPaing, M.M., et al. (2002). J. Biol. Chem. 277, 1292-1300. Sau-Ching Wu, S.-C., et al. (2002). Appl. Envir. Microbiol. 68, 3261-3269.


Product # Description Pkg. Size Price34026 ABTS 50 tablets (10 mg/tablet) $ 8137615 1-Step™ ABTS 250 ml (Ready-to-use) $ 70

52

CHOOSING A SUBSTRATEColorimetric Substrates for HRP

KineticMeasurement Absorbance

Description Highlights Sensitivity Possible Maximum Color Product #1-Step™ ABTS • One component Low Yes 405 nm Green 37615

• Ready to use• Excellent choice when maximum sensitivities are not required• Slow reaction that can be easily followed with a kinetic reader

ABTS Tablets Low Yes 405 nm Green 34026OPD Tablets • OPD can be easily dissolved in a substrate buffer such as High No 492 nm Yellow- 34006

Stable Peroxide Substrate Buffer (10X) (Product # 34062) orangeOPD Powder High No 492 nm Yellow- 34005

orangeTMB Substrate Kit • Highest sensitivity Very High No 450 nm Yellow 34021

• Easy to use• Results in seconds• No DMF or DMSO in reagent

1-Step™ Ultra • One component Very High No 450 nm Yellow 34028TMB-ELISA • Ready to use

• Highest sensitivity• No DMF or DMSO in the reagent

1-Step™ Turbo TMB • One component High No 450 nm Yellow 34022• Ready to use• No DMF or DMSO in the reagent

1-Step™ Slow TMB • One component Medium Yes 450 nm Yellow 34024• Ready to use• No DMF or DMSO in the reagent


TMB

TMB is a chromogen thatyields a blue color (measur-able at 370 nm or 652 nm)when oxidized with hydrogenperoxide (catalyzed by HRP).The color then changes toyellow (measured at 450 nm)upon addition of sulfuric orphosphoric acid to stop the

reaction. TMB is very sensitive and more quickly oxidized thanother HRP substrates, resulting in faster color development.

The 1-Step™ TMB Substrates are one-component substrates thatrequire no preparation before use. Unlike other commerciallyavailable substrates, these products contain no DMF or DMSO.There are three formulations that differ primarily in their sensi-tivities. 1-Step™ Slow TMB is intermediate in sensitivity – idealfor kinetic readings. The sensitivity of the 1-Step™ Turbo TMBcompares to that of OPD used at approximately 1 mg/ml. 1-Step™

Ultra TMB-ELISA produces the highest signal:noise ratio andsensitivity in the picogram range.

Highlights:• Ready-to-use single component• No hydrogen peroxide required• No filtering required• Noncarcinogenic• Various sensitivities to suit any assay

Figure 24. Comparison of sensitivities of Turbo TMB, Slow TMB and ABTS.

Figure 25. 1-Step™ Ultra TMB-ELISA provides more signal than otherTMB substrates.

Figure 26. 1-Step™ Ultra TMB-ELISA produces higher signal:noise (S/N)ratios than other TMB substrates.

ReferencesHong, P.W., et al. (2002). J. Virol. 76, 12855-12865.Murphy, M.B., et al. (2003). Nucleic Acids Res. 31, e110. Su, S.V., et al. (2004). J. Biol. Chem. In press.Tek, V. and Zolkiewski, M. (2002). Protein Sci. 11, 1192-1198. Thomas, P.E., et al. (1976). Anal. Biochem. 75, 168-176.Weimer, B.C., et al, (2001). Appl. Envir. Microbiol. 67, 1300-1307. Wu, S.-C. and Wong, S.-L. (2002). Appl. Envir. Microbiol. 68, 1102-1108.


Product # Description Pkg. Size Price34024 1-Step™ Slow TMB 250 ml $ 6634022 1-Step™ Turbo TMB 250 ml $ 9134028 1-Step™ Ultra TMB-ELISA 250 ml $10134021 TMB Substrate Kit Kit $102

Includes: Peroxidase Substrate (TMB) 200 mlPeroxide Solution (H202) 200 ml

S/N

Ratio

TMB Sources Used

9.0

7.5

6.0

4.5

3.0

1.5

0

Pierce

1-Step

™

Ultra TM

B – EL

ISA

Compe

titor B

Compe

titor D

Compe

titor N

Compe

titor K

Compe

titor I

Compe

titor M

Abso

rban

ce a

t 450

nm

Concentration of Human IFN Gamma (pg/ml)50 100 150 200 250 300 350 400 450 500

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0

Pierce 1-Step™ Ultra TMB - ELISACompetitor B

Competitor DCompetitor ICompetitor K

Competitor MCompetitor N

0

ABTSSlow TMBTurbo TMB

1.5

1.0

0.5

0.0

Abso

rban

ce a

t 450

nm

(TM

B)Ab

sorb

ance

at 4

10 n

m (A

BTS)

Dilution of HRP Conjugate(from 1 mg/ml stock)

1.56 x 10-5 6.25 x 10-5 1.25 x 10-4 2.5 x 10-4

3.13 x 10-5

54


PNPPDetection of alkaline phosphatase label in ELISA applications.

PNPP (p -Nitrophenyl Phosphate, Disodium Salt) is a widelyused substrate for detecting alkaline phosphatase in ELISAapplications.1 When alkaline phosphatase and PNPP arereacted, a yellow water-soluble reaction product is formed.This product absorbs light at 405 nm. Pierce offers PNPP infour formats. PNPP is available either as a crystalline powderor 5 mg tablets. Also available is the Phosphatase SubstrateKit, which contains PNPP tablets and a Diethanolamine Bufferto yield more than one liter of substrate. The DiethanolamineSubstrate Buffer (Product # 34064) is also provided individuallyas a 5X concentrate. The Pierce formulation has an optimaland consistent pH and it is stable even at a 1X concentration.

Until recently, the method for preparing PNPP solutions requireddissolving the powder or tablets in buffer and then diluting thesolution to the desired concentration. Substrate instability madeit necessary to prepare it on a daily basis. Pierce 1-Step™ PNPPcircumvents this time-consuming and variable-introducingstep by providing a single-component PNPP substrate. Thissubstrate is stable for 12 months at 2-8°C and can be stoppedwith conventional methods.

Reference1. Snyder, S.L., et al. (1972). Biochim. Biophys. Acta 258, 178-187.Bosque, P.J., et al. (2002) Proc. Natl. Acad. Sci. 99, 3812-3817. Jan, J.-T., et al. (2000) J. Virol. 74, 8680-8691.


Product # Description Pkg. Size Price34045 PNPP 25 g powder $10034047 PNPP Tablets 105 tablets $102

(5 mg/tablet)37620 Phosphatase Substrate Kit Kit $135

Sufficient reagents for 1.05 liters of substrate.Includes: Diethanolamine Buffer 225 ml

PNPP Tablets 105 tablets(5 mg/tablet)

37621 1-Step™ PNPP 100 ml $ 77

Alkaline Phosphatase Substrates

Alkaline phosphatase, a 140 kDa protein that is generallyisolated from calf intestine, catalyzes the hydrolysis of phosphategroups from a substrate molecule, resulting in a colored orfluorescent product or the release of light as a byproduct. APhas optimal enzymatic activity at a basic pH (pH 8-10) and canbe inhibited by cyanides, arsenate, inorganic phosphate anddivalent cation chelators, such as EDTA. As a label for ELISA,AP offers a distinct advantage over other enzymes. Since itsreaction rate remains linear, detection sensitivity can be improvedby simply allowing a reaction to proceed for a longer time period.Due to the large size of AP, it is very sensitive to freeze/thawand should be aliquotted and stored at 4°C.

The most common ELISA substrate for alkaline phosphataseis the chromogen p -nitrophenyl phosphate (PNPP), which isavailable in several formats. PNPP yields a yellow reactionproduct that is water-soluble and absorbs light at 405 nm.The 1-Step™ PNPP Substrate (Product # 37621) offers theconvenience of a ready-to-use reagent with similar sensitivityto the two-component kit. The Phosphatase Substrate Kit(Product # 37620) includes PNPP tablets and diethanolaminebuffer (5X). PNPP is also available as 25 g of powder (Product# 34045) or in tablet form (Product # 34047). DiethanolamineSubstrate Buffer (Product # 34064) is a convenient, ready-to-useformulation sold as a 5X concentrate with an optimal andconsistent pH. Soluble ELISA substrates for AP are summarizedin Table 7, on page 46.

Kinetic Measurement Absorbance Description Features/Benefits Sensitivity Possible Maximum Color Product #1-Step™ PNPP • One component High Yes 405 nm Yellow 37621

• Ready to use• Stable for 12 months• The easiest-to-use PNPP substrate available

Phosphatase • Ideal for ELISA High Yes 405 nm Yellow 37620Substrate Kit • Easy to use

• Minimal assay-to-assay variability• Low background

PNPP Tablets High Yes 405 nm Yellow 34047PNPP Powder High Yes 405 nm Yellow 34045

Substrate BuffersSubstrate buffers for alkaline phosphatase and horseradish peroxidase.

Buffered stable peroxide is used by researchers who prefer tomake their own HRP-ELISA substrates. This is done easily byadding a chromogen of choice to the buffer. The Stable PeroxideSubstrate Buffer available from Pierce has a long shelf life(12 months after receipt), and is provided as a 10X concentrate.A total volume of 1,000 ml can be made from one 100 ml bottleof concentrate. If you use 10 ml of substrate per plate, you canmake substrate for 100 plates from only one bottle of StablePeroxide Substrate Buffer. Diethanolamine Substrate Bufferis used with soluble alkaline phosphatase substrates such asPNPP. Our formulation is convenient, ready-to-use and reducesthe possibility of assay-to-assay variability. DiethanolamineSubstrate Buffer is sold as 5X concentrate with an optimal,consistent pH. It is stable even at a 1X concentration.

Highlights:• Ready-to-use• Minimize assay-to-assay variability

ReferencesLouis, H., et al. (2000) J. Histochem. Cytochem. 48, 499-508. Neisewander, J.L., et al. (2000) J. Neurosci. 20, 798-805. Pifer, J., et al. (2002) J. Immunol. 169, 1372-1378.


Product # Description Pkg. Size Price34062 Stable Peroxide Substrate Buffer (10X) 100 ml $ 4034064 Diethanolamine Substrate Buffer (5X) 225 ml $ 47

ONPG Colorimetric β-Galactosidase Soluble SubstrateFor detection of β-Galactosidase label in ELISA applications.

β-Galactosidase Soluble Colorimetric SubstrateKineticMeasurement Absorbance

Description Highlights Sensitivity Possible Maximum Color Product #ONPG • Forms a completely soluble product when reacted with β-Galactosidase High Yes 420 nm Yellow 34055

• High extinction coefficient at 405 nm• Yields a yellow product easily detectable in the visual range

When using β-Galactosidase as the label for proteins in ELISAstudies, a wide variety of substrates are available, includingo -nitrophenyl-β-D-galactopyranoside (ONPG), naphthol-AS-BI-β-D-galactopyranoside (Nap-Gal) and 4-Methyl-umbelliferyl-β-D-galactopyranoside (Mum-Gal). However, it is important tochoose a substrate with adequate solubility, that uses readilyavailable equipment and that gives a significant reading overthe background. ONPG is a superior β-Galactosidase substrateoption.1 The product formed is completely soluble and has ahigh extinction coefficient at 405 nm. The substrate yields ayellow product that is easily detectable in the visual range afterstopping the reaction with 1 M sodium carbonate.

Reference1. Craven, G.R., et al. (1965). J. Biol. Chem. 240, 2468-2477.


Product # Description Pkg. Size Price34055 ONPG 5 g powder $ 63


Easy-Titer® ELIFA System

The Easy-Titer® ELIFA System offers faster ELISA results byoverriding limiting diffusion, which is the rate limiting step inELISA.1,2 Limiting diffusion is a phenomenon in which solublereactants are depleted near the reaction surface, and the rateof reaction is limited by diffusion of new reactants to the site.3The enzyme-linked immunoflow assay (ELIFA) system actuallyreduces one-hour incubations to just a five-minute filtration step.A vacuum pulls substrates through a transfer membrane thathas complementary ligands or enzymes bound to it. Cannulasprecisely and quantitatively transfer unbound substrate to thecollection chamber and transfer colored product into corre-sponding microwells (Figure 27) for analysis in an automatedmicroplate reader. Assay times required for the Easy-Titer® ELIFASystem and a traditional ELISA are compared in Table 8.

Highlights:• Allows complete ELISA-type assays to be performed in just

25 minutes• Offers greater sensitivity because transfer membranes bind

more protein than plastic surfaces• 96-well arrangement is compatible with 96-well microplates

and multi-channel pipettes• Most wash steps are eliminated, further reducing assay time• No cross-contamination between samples because of tight seals• Results are easily quantitated

Figure 27. Easy-Titer® ELIFA System. Colored product is transferred tomicroplate wells for an ELISA or precipitated on the membrane for a dot blot.

Table 8. Comparison of assay times for Easy-Titer® ELIFA System and standard ELISA.

Faster Than ELISA Easy-Titer® System ELISAAdsorb antibody 5 minutes 1 hourBlock empty sites 5 minutes 30-60 minutesBind antigen 5 minutes 1 hourAntibody-enzyme 5 minutes 1 hourProduce color 5 minutes 10-30 minutesTotal Assay Time 25 minutes 3.6-4.5 hours

References1. Engvall, E. and Perlmann, P.O. (1971). Immunochemistry 8, 871-875.2. Palomäki, P. (1991). J. Immunol. Method 145, 55-63.3. Rao, P.N. and Taraporewala, I.B. (1992). Steroids 57, 154-161.4. Coons, A.A., et al. (1942). J. Immunol. 45, 159-170.5. Weller, T.H. and Coons, A.H. (1954). Proc. Soc. Exp. Biol. (New York) 86, 789-794.6. Schwab, C. and Bosshard, H.R. (1992). J. Immunol. Method 147, 125-134.7. Stenberg, M. and Nygren, H. (1988). J. Immunol. Method 113, 3-15.8. Valkirs, G.E. and Barton, R. (1985). Clin. Chem. 31, 1427-1431.9. Ijsselmuiden, O.E., et al. (1989). J. Immunol. Method 119, 35-43.

Collection chamber

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GasketsMembrane

Sample application plate

Sample well

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ELIFASYSTEM


Easy-Titer® ELIFA SystemReduces each step to a five-minute filtration!

Highlights:• Effective ELISA device

allows immunoassaysto be completed in just25 minutes (each stepis reduced to a 5-minutefiltration)

• Ideal for ELISA-typeimmunoassays, dot blotsand nucleic acid blots

• Binding kinetics of any ELISA step performed using Easy-Titer®

System overrides limiting diffusion• Greater sensitivity because transfer membranes bind more

protein than plastic surfaces• 96-well arrangement is compatible with 96-well microplates

and multichannel pipettes• Most wash steps are eliminated, further reducing assay time• Cross-talk between samples is eliminated due to the use of

tight seals• Results are quantitated easily by simply drawing color solution

into a microplate and reading using a standard ELISA reader• Use insoluble or precipitating substrates to create dot blot results

Figure 28. A checkerboard titration is a single experiment in which theconcentration of two components is varied in a way to visualize the bestsignal:noise ratio conditions.

References1. Paffard, S.M., et al. (1996). J. Immunol. Method 192, 133-136.Liu, Z.-H., et al. (2001). Proc. Natl. Acad. Sci. 98, 7289-7294. Menalled, L.B., et al. (2002). J. Neurosci. 22, 8266-8276.


Product # Description Pkg. Size Price77000 Easy-Titer® ELIFA System* $1,011

Includes: Sample application plateTop silicone gasketMembrane support plate with bottom silicone gasket and 96 cannulas

Vacuum chamber with o-ringThumbscrews 4One-way valves 4Valve/tubing adapters 4Tubing 4 ftExtra cannulas 10

77015 Biodyne® A Nylon Membranes 25/pkg. $ 26577016 Biodyne® B Nylon Membranes 25/pkg. $ 19077070 Easy-Titer® Replacement Cannulas 100/pkg. $ 5777071 Easy-Titer® Replacement 4/pkg. $ 104

96-Well Clear Silicone Gaskets* The Easy-Titer ® ELIFA System is protected by U.S. patent # 5,219,528.

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BULK AND CUSTOMOFFERINGS

Coated Microplates (96-well or 384-well)

Partner with Pierce!Pierce can also help you commercialize your ELISA kit. We have the expertise, manufacturing capability and ISO 9001 certificationto help you launch a new dimension to your business. Our scientific staff will work with your research team to implement world-classproject management and meet your deadlines for a new product launch. Partner with Pierce to reach the next level of commercialsuccess for your company.

* Minimum quantities apply based on largest catalog package size available. Call for more information.

Need more product than our catalog package size offers?

Contact the Bulk & Custom Products Department at Pierce or your local distributor torequest a price quote that will satisfy your large-scale needs. All of our popular ELISAproducts are available in bulk quantities* and in special package sizes. Delivery can bearranged to meet your schedule. Pierce can bring all of these critical componentstogether to help you develop your own ELISAs on a grand scale:

Blocking Buffers• Serum-based Blockers • Non-serum Blockers• Milk-based Buffers

Immunoassay Buffers• Sample Preparation Buffers • Wash Buffers• Assay Reagent Diluents • Stop Solutions

Soluble Substrates• Colorimetric • Chemiluminescent• Fluorescent

Enzyme Conjugate Stabilizer

Immunoassay Conjugates• Antibody-enzyme Conjugates • NeutrAvidin™-enzyme Conjugates• Biotinylated Antibody Conjugates • Protein A, G, L or A/G-enzyme Conjugates• Streptavidin-enzyme Conjugates • Nickel-chelate Enzyme Conjugate

800-874-3723 • 815-968-0747 • www.piercenet.com 59BULKAND CUSTOMOFFERINGS

ChoiceCoat® Custom Microplate Packaging OptionsPierce can provide the appropriate packaging for your custommicroplates, based upon your usage. Coated microplates canbe packaged for large-screening applications (i.e., packaged in25 plates ready for stacking) or for inclusion in a final kit forresale (i.e., single-pouch packages). Stability testing forcustom microplates can be determined if long-term storage iscritical for your application. Scheduled shipments can beestablished easily to make the microplates available when youneed them most.

Minimum Quantities for ChoiceCoat® Custom Microplate-coating Service Get started creating your own custom microplate with as fewas 500 (96-well) microplates or 200 (384-well) microplates.Pierce will provide five coated microplates at no charge fortesting purposes before we ship any lot of ChoiceCoat®

Custom Coated Plates to you. Pierce will work closely withyou to develop the appropriate quality assurance tests so thatwe provide the most consistent product to meet your needs.

In the United States, contact the Pierce Bulk & Custom SalesDepartment at 1-800-874-3723 or 815-968-0747. Outsidethe United States, contact your local Perbio Science office orPierce distributor for a FREE ChoiceCoat® Consultation.

Partner with Pierce to discover the best choices!

The choice is up to you!

The ChoiceCoat® Custom Microplate-coating Service from PiercePierce has been coating plates for more than 10 years. Putthis experience and expertise to work for you!

Pierce can process up to 7,500 microplates per day and hasdeveloped novel chemistries and coatings for a number ofapplications. Following are just a few of the applications inwhich Pierce custom-coated microplates have been effectivein generating valuable results.

• Competitive LigandBinding Assays

• Oligonucleotide Binding Assays • Protease Assays• Kinase and Phosphatase Assays• Sandwich ELISAs• Pull-down Assays and Immunoprecipitation

ChoiceCoat® Microplate Coating Options Available

Microplate Type/Manufacturer Coat Protein/Ligand Blocking BufferBlack (96- or 384-well) Antibodies SuperBlock® BlockerWhite (96- or 384-well) Bacterial Fusion Proteins StartingBlock™ BlockerClear (96- or 384-well) Oligonucleotides or Peptides Purified CaseinClear bottom, Black Biological Polymers BSAClear bottom, White Metal Chelates SerumYour Microplate Your Ligand Your BlockerNote: Covalent chemistries also an option!

800-874-3723 • 815-968-0747 • www.piercenet.com60

RECOMMENDED READING

Antibodies: A Laboratory ManualMore than 700 pages of valuable information about antibodies and antibody production.

This valuable manual contains 726pages of information about the theory,production and use of monoclonal andpolyclonal antibodies. Oversized printand colorful graphics make it easy toread and understand.


Product # Description Price15051 Antibodies: A Laboratory Manual $132

Harlow, E. and Lane, D.,Published by Cold Spring Harbor Laboratory, 1988,726 pages, Softcover

ELISA Theory and PracticeBasic principles and techniques.

The ELISA Guidebook presents theprinciples of the assay technique calledenzyme linked immunosorbent assay.The book gives detailed descriptions ofthe basic ELISA principles and techniquesthat will aid both novice and experiencedresearchers in developing their ownrapid and accurate assays.

This 421-page book features thefollowing range of topics:

• Direct, indirect and competitive ELISA• Capture ELISA on solid-phase• Basic immunology• Stages in ELISA• Theoretical considerations• Practical exercises• Immunochemical techniques


Product # Description Price15056 The ELISA Guidebook $100

Crowther, J.R., Published by Humana Press, Inc., 2001,421 pages, Softcover

Protein MethodsTreats practical protein-related issues that most other books do not address.

This expanded second edition providesstep-by-step explanations of basicmethods for extracting, handling,storing, measuring, solubilizing andconcentrating proteins.


Product # Description Price20001 Protein Methods $ 78

Bollag, D.M., et al., Published by Wiley-Liss, Inc., 1996,400 pages, Comb-bound

RECOMMENDEDREADING 61

Protein Assay Technical Handbook

The 38-page Protein Assay TechnicalHandbook is a helpful guide to choosingthe best protein assay and standard for asample. It contains abundant informationon the most widely used protein assaymethods (BCA™, Lowry and BradfordAssays), including the principle behindeach protein assay and its uniqueadvantages and disadvantages. The

handbook also includes methods for detection specific proteintypes such as histidine-tagged proteins, antibodies and proteases.


Product # Description Price1601004 Protein Assay Technical Handbook FREE

Western Blotting Handbook and Troubleshooting Guide

This 52-page handbook containsfull-length and abbreviated Westernblotting protocols, tips on antibody andblocking buffer optimization, a chemilu-minescent substrate selection guide, aprocedure for stripping and reprobingWestern blots, tips on clearer filmdevelopment, and other information thatwill be useful to experts and first-timers

alike. Learn more about four new Western blotting products thatcan improve your blot’s signal:noise ratio: Restore™ Western BlotStripping Buffer, Qentix™ Western Blot Signal Enhancer, Erase-It®

Background Eliminator and StartingBlock™ Blocking Buffers.


Product # Description Price1600990 Western Blotting Brochure FREE

Antibody Technical Handbook

This new 68-page handbook helps youchoose the best methods to produce,purify, fragment and label antibodies.Topics include basic immunology,carrier proteins, adjuvants, antibodypurification methods, antibody frag-mentation with proteases, and labelingantibodies with a variety of tags (e.g.,biotin, fluorophores, enzymes, iodine)for purification or detection.


Product # Description Price1601092 Antibody Technical Handbook FREE

Sorry, books are nonreturnable.

G r a s p t h e P r o t e o m e ™

Tel: 815-968-0747 or 800-874-3723 • Fax: 815-968-7316Customer Assistance E-mail: [email protected]

Outside the United States, visit our web site or call 815-968-0747 to locate your local Perbio Science branch office (below) or distributor

© Pierce Biotechnology, Inc., 2004. Printed in the U.S.A. Pierce products are supplied for laboratory or manufacturing applications only.Prices listed herein are accurate at time of printing. CyDye™, Cy3™ and Cy5™ are registered trademarks of Amersham Biosciences. Tween® and Brij® are registered trademarks of ICI Americas.

Kathon® and Triton® are registered trademarks of Rohm & Hass Co. Biodyne® is a trademark of Pall Corporation. Texas Red® is a trademark of Molecular Probes, Inc.

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SuperSignal® ELISA Substrates• Strong, sensitive signal – clear detection

in the femtogram range• Low noise – no auto-generated back-

ground like many fluorescent systems• Immediate signal generation – no

waiting for detection• Stable light output – no drop in signal

for up to 30 minutes

StartingBlock™ Buffers• Limits background when others don’t –

guaranteed serum protein- and biotin-free formulations

• Does not cross-react with rabbit anti-bodies – a common problem withother blockers

• “No-wait” blocking – just pipette and remove

• Convenient – buffers are available withor without Tween®-20 Detergent added

Reacti-Bind™ Coated Plates• Pre-coated plates – for binding, anti-

bodies, fusion proteins and biotin orfor covalently binding targets

• Selective – plates bind only the targetligand, not unwanted, noise-producingmolecules

• Convenient normal- and high-bindingversions are available

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Turn down the noise!Three ways to get the highest signal:noise ratio possible.

Pump up the volume!

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