Millipore, Advancing Life Science Together, SNAP i.d., Immobilon, Amicon, and Ultracel are registered trademarks of Millipore Corporation. ReNcell, PureProteome, Luminata, Bløk, ReBlot, ChemiLucent, and the M mark are trademarks of Millipore Corporation. Qdot is a trademark of Quantum Dot Corporation. Kodak is a registered trademark of Eastman Kodak Company. Lit. No. PB1033EN00 Printed in the USA 03/10 LS-SBU-10-02831 ©2010 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

Immobilon Transfer MembranesDescription Size Qty/Pk Catalogue No.

Immobilon P: PVDF 0.45 µm 7 x 8.4 cm 50/pk IPVH07850

26.5 cm x 3.75 m 1 roll IPVH00010

Immobilon PSQ: PVDF 0.2 µm 7 x 8.4 cm 50/pk ISEQ07850

26.5 cm x 3.75 m 1 roll ISEQ00010

Immobilon FL: PVDF 0.45 µm 7 x 8.4 cm 10/pk IPFL07810

26.5 cm x 3.75 m 1 roll IPFL00010

SNAP i.d. System

Description Components Qty/Pk Catalogue No.

SNAP i.d. Protein Detection System WBAVDBASE

SNAP i.d Consumables and Accessories Single Blot Holder 30/pk WBAVDBH01

Double Blot Holder 30/pk WBAVDBH02

Triple Blot Holder 20/pk WBAVDBH03

Antibody Collection Tray 20/pk WBAVDABTR

SNAP i.d. Blot Roller 1/pk WBAVDR0LL

Bløk Noise-Canceling ReagentsDescription Detection Method Qty/Pk Catalogue No.

Bløk-CH Reagent Chemiluminescence Detection 500 mL/bottle WBAVDCH01

Bløk-FL Reagent Fluorescence Detection 500 mL/bottle WBAVDFL01

Bløk-PO Reagent Phosphoprotein Detection 500 mL/bottle WBAVDP001

Luminata Western HRP SubstratesDescription Qty/Pk Catalogue No.

Luminata Classico Western HRP Substrate

500 mL WBLUC0500

Luminata Crescendo Western HRP Substrate

500 mL WBLUR0500

Luminata Forte Western HRP Substrate

500 mL WBLUF0500

Western Blotting Enhancing ReagentsDescription Qty/Pk Catalogue No.

ChemiLucent Plus Western Blot Enhancing Kit

1 kit 2650

ReBlot Plus Mild Antibody Stripping Solution, 10x

50 mL 2502

ReBlot Plus Strong Antibody Stripping Solution, 10x

50 mL 2504

ORdERINg INfORMATION

Western Blotting ToolsPublication Quality Westerns in Minuteswith Millipore’s Pre-optimized Products

TO PLACE AN ORdER In the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)

In Europe, please call Customer Service:

France: 0825.045.645 • Spain: 901.516.645 Option 1 • Germany: 01805.045.645 • Italy: 848.845.645 • UK: 0870.900.46.45

For other countries across Europe and the world, please visit www.millipore.com/offices.

For Technical Service, please visit www.millipore.com/techservice.

www.millipore.com/WBtools

3 3

One of the greatest challenges in advancing research is obtaining

consistent, quality results. In Western blotting, the most important

factor in determining the success of experiments is the quality of

resources used, including the protein extraction kit, transfer membrane

and reagents. Millipore offers an array of Western blotting products

that are pre-optimized to work synergistically, providing strong specific

signals and low background to help you quickly produce publication

quality results.

Ready, set, publish! At each step in the Western blotting workflow,

choose the Millipore product with unique advantages that will drive

your research forward.

Millipore’s Western blotting portfolio delivers the highest quality in the shortest time, setting a new pace for discovery.

ZERO TO PuBLICATION QuALITy WESTERNS IN Protein Extraction &

Sample Preparation Protein extraction and purification represent the first of many challenges in obtaining intact, active proteins.

Millipore’s quality reagents unite superior performance with speed to reduce exposure of proteins to unfavorable

conditions, leading to more stable, intact proteins for downstream analysis.

Extraction Kits and Protease InhibitorsProtein stability is fundamental to all aspects of protein research, including analysis by Western blotting. Combine our gentle protein extraction kits with protease inhibitors to obtain stabilized, intact, and active proteins.

Description Qty/Pk Catalogue No.

Compartment Protein Extraction Kit 1 kit 2145

Total Protein Extraction Kit 1 kit 2140

Nuclear Extraction Kit 100 assays 2900

RIPA Lysis Buffer, 10X 100 mL 20-188

Protease Inhibitor Cocktail 1 vial 20-201

Chymostatin 100 mg EI6

Leupeptin 100 mg EI8

Pepstatin A 100 mg EI10

Affinity PurificationPurify your protein with PureProteome magnetic beads or agarose beads. PureProteome magnetic beads ensure fast, effective isolation of proteins without sample loss and are available in nickel, protein A, or protein G formats.

Description Qty/Pk Catalogue No.

PureProteome Nickel Magnetic Beads 10 mL LSKMAGH10

PureProteome Protein A Magnetic Beads 10 mL LSKMAGA10

PureProteome Protein G Magnetic Beads 10 mL LSKMAGG10

Protein A Agarose, fast flow 10 mL 16-156

Protein G Agarose, fast flow 10 mL 16-266

Streptavidin Agarose Conjugate 10 mL 16-126

Buffer Exchange and ConcentrationSimultaneously concentrate and desalt your samples with Amicon Ultra centrifugal filters. Their unparalleled rapid and reproducible performance minimizes protein exposure to harsh buffers.

Description Qty/Pk Catalogue No.

Amicon Ultra - 0.5 mL Filters* 24/pk UFC501024

96/pk UFC501096

Amicon Ultra - 4 mL Filters* 24/pk UFC801024

96/pk UFC801096

Amicon Ultra - 15 mL Filters* 24/pk UFC901024

96/pk UFC901096

*10,000 MWCO. For additional MWCOs, visit www.millipore.com or contact Technical Service.

Protein Extraction & Preparation Blocking detection

Electrophoresis & Transfer

Antibody Incubation,

Washing

Gentle protein extraction kits, pg. 3

Rapid protein isolation with PureProteome™ magnetic beads, pg. 3

Fast, effective concentration with Amicon® Ultra centrifugal filters, pg. 3

High protein binding with Immobilon® membranes, pg. 4

22-minute immunodetection with SNAP i.d.® protein detection system, pg. 6

Protein-free Bløk™ noise-cancelling reagents, pg. 8

SNAP i.d. protein detection system,pg. 6

Optimized antibodies for the SNAP i.d. detection system, pg. 9

Premixed Luminata™ Western HRP substrates for stronger signals, pg. 10

Protein Extraction & Preparation

22MINuTES!

3 3

One of the greatest challenges in advancing research is obtaining

consistent, quality results. In Western blotting, the most important

factor in determining the success of experiments is the quality of

resources used, including the protein extraction kit, transfer membrane

and reagents. Millipore offers an array of Western blotting products

that are pre-optimized to work synergistically, providing strong specific

signals and low background to help you quickly produce publication

quality results.

Ready, set, publish! At each step in the Western blotting workflow,

choose the Millipore product with unique advantages that will drive

your research forward.

Millipore’s Western blotting portfolio delivers the highest quality in the shortest time, setting a new pace for discovery.

ZERO TO PuBLICATION QuALITy WESTERNS IN Protein Extraction &

Sample Preparation Protein extraction and purification represent the first of many challenges in obtaining intact, active proteins.

Millipore’s quality reagents unite superior performance with speed to reduce exposure of proteins to unfavorable

conditions, leading to more stable, intact proteins for downstream analysis.

Extraction Kits and Protease InhibitorsProtein stability is fundamental to all aspects of protein research, including analysis by Western blotting. Combine our gentle protein extraction kits with protease inhibitors to obtain stabilized, intact, and active proteins.

Description Qty/Pk Catalogue No.

Compartment Protein Extraction Kit 1 kit 2145

Total Protein Extraction Kit 1 kit 2140

Nuclear Extraction Kit 100 assays 2900

RIPA Lysis Buffer, 10X 100 mL 20-188

Protease Inhibitor Cocktail 1 vial 20-201

Chymostatin 100 mg EI6

Leupeptin 100 mg EI8

Pepstatin A 100 mg EI10

Affinity PurificationPurify your protein with PureProteome magnetic beads or agarose beads. PureProteome magnetic beads ensure fast, effective isolation of proteins without sample loss and are available in nickel, protein A, or protein G formats.

Description Qty/Pk Catalogue No.

PureProteome Nickel Magnetic Beads 10 mL LSKMAGH10

PureProteome Protein A Magnetic Beads 10 mL LSKMAGA10

PureProteome Protein G Magnetic Beads 10 mL LSKMAGG10

Protein A Agarose, fast flow 10 mL 16-156

Protein G Agarose, fast flow 10 mL 16-266

Streptavidin Agarose Conjugate 10 mL 16-126

Buffer Exchange and ConcentrationSimultaneously concentrate and desalt your samples with Amicon Ultra centrifugal filters. Their unparalleled rapid and reproducible performance minimizes protein exposure to harsh buffers.

Description Qty/Pk Catalogue No.

Amicon Ultra - 0.5 mL Filters* 24/pk UFC501024

96/pk UFC501096

Amicon Ultra - 4 mL Filters* 24/pk UFC801024

96/pk UFC801096

Amicon Ultra - 15 mL Filters* 24/pk UFC901024

96/pk UFC901096

*10,000 MWCO. For additional MWCOs, visit www.millipore.com or contact Technical Service.

Protein Extraction & Preparation Blocking detection

Electrophoresis & Transfer

Antibody Incubation,

Washing

Gentle protein extraction kits, pg. 3

Rapid protein isolation with PureProteome™ magnetic beads, pg. 3

Fast, effective concentration with Amicon® Ultra centrifugal filters, pg. 3

High protein binding with Immobilon® membranes, pg. 4

22-minute immunodetection with SNAP i.d.® protein detection system, pg. 6

Protein-free Bløk™ noise-cancelling reagents, pg. 8

SNAP i.d. protein detection system,pg. 6

Optimized antibodies for the SNAP i.d. detection system, pg. 9

Premixed Luminata™ Western HRP substrates for stronger signals, pg. 10

Protein Extraction & Preparation

22MINuTES!

4 5

Immobilon Western Blotting Transfer Membranes

The signal intensity in a Western blot is highly dependent on the

protein’s density after a Western transfer. When an

insufficient amount of protein is bound to the

membrane, a strong specific signal can be very

difficult to obtain. For that reason, the quality of

the Western blotting transfer membrane

is essential to obtaining high signal-

to-noise ratios and clear experimental

results.

The PVDF Immobilon membranes provide high

protein binding capacity resulting in strong signals.

Each of the Immobilon membranes has been optimized for a

different protein blotting application.

Immobilon-Ptransfer membrane

Immobilon-PSQ

transfer membraneImmobilon-FLtransfer membrane

description Optimized to bind proteins transferred from a variety of gel matrices

Uniform pore structure results in superior binding of proteins with MW <20 kDa

Optimized for fluorescence immunodetection applications

Composition PVDF PVDF PVDF

Pore size 0.45 µm 0.2 µm 0.45 µm

Phobicity Hydrophobic Hydrophobic Hydrophobic

Applications • Western blotting• Binding assays• Amino acid analysis• N-terminal protein sequencing• Dot/slot blotting• Glycoprotein visualization• Lipopolysaccharide analysis• Mass spectrometry

• Low molecular weight Western blotting• Amino acid analysis• Mass spectrometry• N-terminal protein sequencing

• Western blotting• Dot/slot blotting• Fluorescence immunodetection

detection methods • Chromogenic• Chemiluminescent• Radioactive

• Chromogenic• Chemiluminescent• Radioactive

• Fluorescent• Chromogenic• Chemifluorescent• Chemiluminescent

Protein binding capacity Insulin: 160 µg/cm2

BSA: 215 µg/cm2

Goat IgG: 294 µg/cm2

Insulin: 262 µg/cm2

BSA: 340 µg/cm2

Goat IgG: 448 µg/cm2

Insulin: 155 µg/cm2

BSA: 205 µg/cm2

Goat IgG: 300 µg/cm2

Comparison of Immobilon membrane properties

Multiplex detection using fluorescent probes on the Immobilon FL membrane Actin-tubulin assay on Immobilon-FL membrane. Rabbit muscle actin (red) was detected using rabbit anti-actin secondary antibodies and QDot® 655 goat anti-rabbit secondary antibodies. Porcine brain tubulin (green) was detected using mouse anti-tubulin primary and QDot 565 goat anti-mouse secondary antibodies. Sensitivities down to 1 ng were observed on a Kodak® imager. Data provided by Quantum Dot Corporation.

Description Size Qty/Pk Catalogue No.

Immobilon P: PVDF 0.45 µm 7 x 8.4 cm 50/pk IPVH07850

26.5 cm x 3.75 m 1 roll IPVH00010

Immobilon PSQ: PVDF 0.2 µm 7 x 8.4 cm 50/pk ISEQ07850

26.5 cm x 3.75 m 1 roll ISEQ00010

Immobilon FL: PVDF 0.45 µm 7 x 8.4 cm 10/pk IPFL07810

26.5 cm x 3.75 m 1 roll IPFL00010

ORdERINg INfORMATION

For a complete listing of available Immobilon membranes, visit www.millipore.com/immobilonwestern.

Immobilon PSQ membrane prevents the proteins from blowing through the membrane, increasing protein signal

Molecular weight standards (lanes 1 and 3) and calf liver lysate (lanes 2 and 4) were transferred to Immobilon-P or Immobilon-PSQ membranes. A sheet of Immobilon-PSQ membrane was placed behind the primary membranes to capture proteins that passed through (lanes 5 and 6 behind Immobilon-P membrane; lanes 7 and 8 behind Immobilon-PSQ membrane).

200

116976655

3631

21.5

14.4

6

3.5

kDa 1 2

P PSQ PSQ Backup

3 4 5 6 7 8

1 ng1000 ng

1000 ng1 ng

Electrophoresis & Transfer

KEy fEATuRES• High protein binding capacity

• Easy to strip and reprobe

• High tensile strength and flexibility

• Highly resistant to organic solvents

Validated for the SNAP i.d. system

4 5

Immobilon Western Blotting Transfer Membranes

The signal intensity in a Western blot is highly dependent on the

protein’s density after a Western transfer. When an

insufficient amount of protein is bound to the

membrane, a strong specific signal can be very

difficult to obtain. For that reason, the quality of

the Western blotting transfer membrane

is essential to obtaining high signal-

to-noise ratios and clear experimental

results.

The PVDF Immobilon membranes provide high

protein binding capacity resulting in strong signals.

Each of the Immobilon membranes has been optimized for a

different protein blotting application.

Immobilon-Ptransfer membrane

Immobilon-PSQ

transfer membraneImmobilon-FLtransfer membrane

description Optimized to bind proteins transferred from a variety of gel matrices

Uniform pore structure results in superior binding of proteins with MW <20 kDa

Optimized for fluorescence immunodetection applications

Composition PVDF PVDF PVDF

Pore size 0.45 µm 0.2 µm 0.45 µm

Phobicity Hydrophobic Hydrophobic Hydrophobic

Applications • Western blotting• Binding assays• Amino acid analysis• N-terminal protein sequencing• Dot/slot blotting• Glycoprotein visualization• Lipopolysaccharide analysis• Mass spectrometry

• Low molecular weight Western blotting• Amino acid analysis• Mass spectrometry• N-terminal protein sequencing

• Western blotting• Dot/slot blotting• Fluorescence immunodetection

detection methods • Chromogenic• Chemiluminescent• Radioactive

• Chromogenic• Chemiluminescent• Radioactive

• Fluorescent• Chromogenic• Chemifluorescent• Chemiluminescent

Protein binding capacity Insulin: 160 µg/cm2

BSA: 215 µg/cm2

Goat IgG: 294 µg/cm2

Insulin: 262 µg/cm2

BSA: 340 µg/cm2

Goat IgG: 448 µg/cm2

Insulin: 155 µg/cm2

BSA: 205 µg/cm2

Goat IgG: 300 µg/cm2

Comparison of Immobilon membrane properties

Multiplex detection using fluorescent probes on the Immobilon FL membrane Actin-tubulin assay on Immobilon-FL membrane. Rabbit muscle actin (red) was detected using rabbit anti-actin secondary antibodies and QDot® 655 goat anti-rabbit secondary antibodies. Porcine brain tubulin (green) was detected using mouse anti-tubulin primary and QDot 565 goat anti-mouse secondary antibodies. Sensitivities down to 1 ng were observed on a Kodak® imager. Data provided by Quantum Dot Corporation.

Description Size Qty/Pk Catalogue No.

Immobilon P: PVDF 0.45 µm 7 x 8.4 cm 50/pk IPVH07850

26.5 cm x 3.75 m 1 roll IPVH00010

Immobilon PSQ: PVDF 0.2 µm 7 x 8.4 cm 50/pk ISEQ07850

26.5 cm x 3.75 m 1 roll ISEQ00010

Immobilon FL: PVDF 0.45 µm 7 x 8.4 cm 10/pk IPFL07810

26.5 cm x 3.75 m 1 roll IPFL00010

ORdERINg INfORMATION

For a complete listing of available Immobilon membranes, visit www.millipore.com/immobilonwestern.

Immobilon PSQ membrane prevents the proteins from blowing through the membrane, increasing protein signal

Molecular weight standards (lanes 1 and 3) and calf liver lysate (lanes 2 and 4) were transferred to Immobilon-P or Immobilon-PSQ membranes. A sheet of Immobilon-PSQ membrane was placed behind the primary membranes to capture proteins that passed through (lanes 5 and 6 behind Immobilon-P membrane; lanes 7 and 8 behind Immobilon-PSQ membrane).

200

116976655

3631

21.5

14.4

6

3.5

kDa 1 2

P PSQ PSQ Backup

3 4 5 6 7 8

1 ng1000 ng

1000 ng1 ng

Electrophoresis & Transfer

KEy fEATuRES• High protein binding capacity

• Easy to strip and reprobe

• High tensile strength and flexibility

• Highly resistant to organic solvents

Validated for the SNAP i.d. system

6 7

SNAP i.d. Protein detection SystemRapid immunodetection in just 22 minutes!

Western blotting has been the life scientist’s workhorse since its introduction in

1979, and it remains the gold standard in protein detection. Few improvements

were made to this laborious, time-consuming technique, until the SNAP i.d.

protein detection system was introduced in 2008. The SNAP i.d. system

decreases the immunodetection phase of Western blotting to 22 minutes by

using two mechanisms to favor antibody-antigen interaction:

1. The SNAP i.d. protocol uses higher antibody (Ab) concentrations

relative to traditional methods, driving antibody (Ab)-antigen (Ag)

complex formation.

2. The SNAP i.d. system uses vacuum suction to pull the antibodies through the membrane, exposing all of the membrane-

embedded target proteins to the antibody. This increases the available antigen concentration, driving the equilibrium to

favor antibody-antigen complex formation.

KEy fEATuRES• 22-minute immunodetection

enabling more experiments

in less time

• Increased antibody-antigen binding

• Superior washes for lower

background

Traditional Western BlotTraditional Western blotting

relies on diffusionThe SNAP i.d. system actively pulls

the reagents through the membrane

SNAP i.d. System

Membrane surface

Entrapped protein

Traditional Western BlotTraditional Western blotting

relies on diffusionThe SNAP i.d. system actively pulls

the reagents through the membrane

SNAP i.d. System

Membrane surface

Entrapped protein

DETECTION OF TRANSFERRINBlots of a serial dilution of human serum were probed with anti-human transferrin followed by anti-sheep rabbit HRP-conjugated IgG secondary antibody (AP147P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).

DETECTION OF VIMENTINBlots of a serial dilution of ReNcell™ CX cell lysates (14, 7, 3 µg) were probed with anti-human vimentin (AB1620, Millipore) followed by anti-goat rabbit HRP-conjugated IgG secondary antibody (AP106P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).

DETECTION OF MAP KINASE 1/2 (ERK 1/2)Blots of a serial dilution of rat liver lysate (6, 3, 1.5 µg) were probed with anti-MAP Kinase 1/2 (06-182, Millipore) followed by anti-goat rabbit HRP-conjugated IgG secondary antibody (AP132P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).

DETECTION OF ADENOVIRUSBlots of a serial dilution of Adenovirus infected HEK cells were probed with anti-Adenovirus clone 20/11 (MAB8052, Millipore) followed by anti-goat mouse HRP-conjugated IgG secondary antibody (AP124P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).

Traditional Western Blot

77 kDa

58 kDa

44 kDa

140 kDa

P=1:80,000S=1:50,000

P=1:20,000S=1:80,000

P=1:1,000S=1:200,000

P=1:5,000S=1:10,000

SNAP i.d. System Conditions

P=1:10,000S=1:20,000

P=1:1,000S=1:10,000

P=1:200S=1:40,000

P=1:1,000S=1:10,000

A.

B.

C.

D.

ORdERINg INfORMATIONDescription Components Qty/Pk Catalogue No.

SNAP i.d. Protein Detection System WBAVDBASE

SNAP i.d Consumables and Accessories Single Blot Holder 30/pk WBAVDBH01Double Blot Holder 30/pk WBAVDBH02Triple Blot Holder 20/pk WBAVDBH03Antibody Collection Tray 20/pk WBAVDABTRSNAP i.d. Blot Roller 1/pk WBAVDR0LL

BlockingAntibody Incubation, Washing

During electrotransfer of proteins from SDS-PAGE gels to membranes, proteins get trapped within the membrane’s 3-dimensional structure. Traditional Western blotting relies on the diffusion of the antibody through the membrane to reach entrapped proteins. This is a very slow and inefficient process. The SNAP i.d. system actively pulls antibodies through the membrane, increasing their exposure to the entrapped proteins and decreasing the required antibody incubation time.

Comparison of Westerns using the SNAP i.d. system and traditional immunoblotting

Traditional Western BlotTraditional Western blotting

relies on diffusionThe SNAP i.d. system actively pulls

the reagents through the membrane

SNAP i.d. System

Membrane surface

Entrapped protein

Traditional Western BlotTraditional Western blotting

relies on diffusionThe SNAP i.d. system actively pulls

the reagents through the membrane

SNAP i.d. System

Membrane surface

Entrapped protein

[Ab] + [Ag] [Ab Ag]

[Ab] + [Ag] [Ab Ag]

•

•

[Ab] + [Ag] [Ab Ag]

[Ab] + [Ag] [Ab Ag]

•

•

6 7

SNAP i.d. Protein detection SystemRapid immunodetection in just 22 minutes!

Western blotting has been the life scientist’s workhorse since its introduction in

1979, and it remains the gold standard in protein detection. Few improvements

were made to this laborious, time-consuming technique, until the SNAP i.d.

protein detection system was introduced in 2008. The SNAP i.d. system

decreases the immunodetection phase of Western blotting to 22 minutes by

using two mechanisms to favor antibody-antigen interaction:

1. The SNAP i.d. protocol uses higher antibody (Ab) concentrations

relative to traditional methods, driving antibody (Ab)-antigen (Ag)

complex formation.

2. The SNAP i.d. system uses vacuum suction to pull the antibodies through the membrane, exposing all of the membrane-

embedded target proteins to the antibody. This increases the available antigen concentration, driving the equilibrium to

favor antibody-antigen complex formation.

KEy fEATuRES• 22-minute immunodetection

enabling more experiments

in less time

• Increased antibody-antigen binding

• Superior washes for lower

background

Traditional Western BlotTraditional Western blotting

relies on diffusionThe SNAP i.d. system actively pulls

the reagents through the membrane

SNAP i.d. System

Membrane surface

Entrapped protein

Traditional Western BlotTraditional Western blotting

relies on diffusionThe SNAP i.d. system actively pulls

the reagents through the membrane

SNAP i.d. System

Membrane surface

Entrapped protein

DETECTION OF TRANSFERRINBlots of a serial dilution of human serum were probed with anti-human transferrin followed by anti-sheep rabbit HRP-conjugated IgG secondary antibody (AP147P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).

DETECTION OF VIMENTINBlots of a serial dilution of ReNcell™ CX cell lysates (14, 7, 3 µg) were probed with anti-human vimentin (AB1620, Millipore) followed by anti-goat rabbit HRP-conjugated IgG secondary antibody (AP106P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).

DETECTION OF MAP KINASE 1/2 (ERK 1/2)Blots of a serial dilution of rat liver lysate (6, 3, 1.5 µg) were probed with anti-MAP Kinase 1/2 (06-182, Millipore) followed by anti-goat rabbit HRP-conjugated IgG secondary antibody (AP132P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).

DETECTION OF ADENOVIRUSBlots of a serial dilution of Adenovirus infected HEK cells were probed with anti-Adenovirus clone 20/11 (MAB8052, Millipore) followed by anti-goat mouse HRP-conjugated IgG secondary antibody (AP124P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore).

Traditional Western Blot

77 kDa

58 kDa

44 kDa

140 kDa

P=1:80,000S=1:50,000

P=1:20,000S=1:80,000

P=1:1,000S=1:200,000

P=1:5,000S=1:10,000

SNAP i.d. System Conditions

P=1:10,000S=1:20,000

P=1:1,000S=1:10,000

P=1:200S=1:40,000

P=1:1,000S=1:10,000

A.

B.

C.

D.

ORdERINg INfORMATIONDescription Components Qty/Pk Catalogue No.

SNAP i.d. Protein Detection System WBAVDBASE

SNAP i.d Consumables and Accessories Single Blot Holder 30/pk WBAVDBH01Double Blot Holder 30/pk WBAVDBH02Triple Blot Holder 20/pk WBAVDBH03Antibody Collection Tray 20/pk WBAVDABTRSNAP i.d. Blot Roller 1/pk WBAVDR0LL

BlockingAntibody Incubation, Washing

During electrotransfer of proteins from SDS-PAGE gels to membranes, proteins get trapped within the membrane’s 3-dimensional structure. Traditional Western blotting relies on the diffusion of the antibody through the membrane to reach entrapped proteins. This is a very slow and inefficient process. The SNAP i.d. system actively pulls antibodies through the membrane, increasing their exposure to the entrapped proteins and decreasing the required antibody incubation time.

Comparison of Westerns using the SNAP i.d. system and traditional immunoblotting

Traditional Western BlotTraditional Western blotting

relies on diffusionThe SNAP i.d. system actively pulls

the reagents through the membrane

SNAP i.d. System

Membrane surface

Entrapped protein

Traditional Western BlotTraditional Western blotting

relies on diffusionThe SNAP i.d. system actively pulls

the reagents through the membrane

SNAP i.d. System

Membrane surface

Entrapped protein

[Ab] + [Ag] [Ab Ag]

[Ab] + [Ag] [Ab Ag]

•

•

[Ab] + [Ag] [Ab Ag]

[Ab] + [Ag] [Ab Ag]

•

•

8 9

Bløk Noise-Cancelling Reagents

Optimized Antibodies for the SNAP i.d. System Obtain fast, reproducible results using antibodies optimized for the SNAP i.d. systemBløk reagents are a family of uniquely formulated protein-free blocking solutions

that provide numerous benefits including:

• Reduced background for chemiluminescent or fluorescent detection.

• Unique formulation for detection of phosphorylated proteins.

• More stable diluent for antibodies than milk.

• Allows chromogenic staining of membranes after immunodetection.

• Engineered for superior reagent flow through SNAP i.d. blot holders.

Target Protein Catalogue No.SNAP i.d. Dilution Factor or Antibody Concentration

Actin, clone C4 MAB1501 1:2000

Akt, phosphotyrosine (Tyr450) 07-1643 1:1000

ATR 09-070 1:500

BRCA-1 clone BC70 05-842 1:500

CAF1 p60, clone SS-53, 1-124 04-1523 1 µg/mL

Catenin, b 06-734 1:200

c-Jun, clone 6E4.4 05-1076 5 µg/mL

CREB (bZIP transcription factor) 06-863 1:200

CyPA (Cyclophilin A) 07-313 1:1000

Endophilin B1 AB10555 1:500-1:1000

FUBP3 07-742 2 µg/mL

G9a (BAT8) 09-071 1 µg/mL

GAPDH MAB374 1:8000

HDAC11 09-827 0.5 µg/mL

Hexim 1 07-955 4 µg/mL

HLX1 09-084 2 µg/mL

IGF2 mRNA-binding protein 2 07-103 4 µg/mL

IGF2 mRNA-binding protein 3 07-104 2 µg/mL

IKKa 07-1007 1 µg/mL

IKKb 07-1008 2-10 µg/mL

IRS1, clone 58-10C-31 05-784R 0.1 µg/mL

JunD 07-1334 1:2000

LSM14A ABE37 1 µg/mL

MAP Kinase 1/2 (Erk-1/2) 06-182 1:500

MYPT1, phosphothreonine (Thr696) ABS45 0.1 µg/mL

NES, Nestin AB5922 1:1000

NEFL, 70 kDa clone DA2 MAB1615 1:200

P38/SAP-K2 05-454 1:600

P 53 AB565 1:1000

PLCg-1, phosphotyrosine (Tyr783) 07-2134 1:1000

PLK1, phosphoserine (Ser137) 07-1348 0.5 µg/mL

Post-immunodetection chromogenic staining of blots When weak signals were obtained in lanes 5 & 6, blots were stained with Coomassie blue following either chemiluminescence (left blots) or fluorescence (right blots) detection to verify equal loading and protein transfer of all lanes.

Lane 1: Molecular weight marker Lane 2: A431 Pervanadate (PVD) stimulated Lane 3: A431 control non stimulated

(fresh samples)Lane 4: A431 EGFR stimulated

(fresh samples)Lane 5: A431 control non stimulated

(old samples)Lane 6: A431 EGFR stimulated

(old samples)

1 2 3 4 5 6 1 2 3 4 5 6

Chemiluminescence detection

Blot stained after detection

1 2 3 4 5 6 1 2 3 4 5 6

Fluorescence detection

Blot stained after detection

ORdERINg INfORMATIONDescription Detection Method Qty/Pk Catalogue No.

Bløk-CH Reagent Chemiluminescence detection 500 mL/bottle WBAVDCH01

Bløk-FL Reagent Fluorescence detection 500 mL/bottle WBAVDFL01

Bløk-PO Reagent Phosphoprotein detection 500 mL/bottle WBAVDP001

BlockingAntibody Incubation

Bløk-PO reagent preserves the phosphorylation state of the protein

Chemiluminescence detection of pERK in EGF-stimulated A431 lysate (10-2.5 µg/lane,12-110, Millipore) using anti-pERK antibody (1:200, 05-797R, Millipore). Bands were detected using Luminata Forte Western HRP substrate (WBLUF0500, Millipore). pERK phosphorylated doublet indicated by arrow. NFDM - Non-fat dry milk

Competitor L Competitor P NFDM Bløk – PO

SNAP i.d. PublicationsMihrshahi R, Barclay AN, Brown MH. J Immunol. 2009 Oct 15; 183(8):4879-86.

Fujimori K, Ueno T, et al. J Biol Chem. 2010 Mar 19; 285(12):8880-6.

Sakane A, Honda K, Sasaki T. Mol Cell Biol. 2010 Feb; 30(4):1077-87.

Many more to be found on www.millipore.com/SnapPub.

Validated for the SNAP i.d. system

For a complete listing, visit the SNAP i.d. Antibody Optimization Reference Guide at www.millipore.com/SNAPab.

8 9

Bløk Noise-Cancelling Reagents

Optimized Antibodies for the SNAP i.d. System Obtain fast, reproducible results using antibodies optimized for the SNAP i.d. systemBløk reagents are a family of uniquely formulated protein-free blocking solutions

that provide numerous benefits including:

• Reduced background for chemiluminescent or fluorescent detection.

• Unique formulation for detection of phosphorylated proteins.

• More stable diluent for antibodies than milk.

• Allows chromogenic staining of membranes after immunodetection.

• Engineered for superior reagent flow through SNAP i.d. blot holders.

Target Protein Catalogue No.SNAP i.d. Dilution Factor or Antibody Concentration

Actin, clone C4 MAB1501 1:2000

Akt, phosphotyrosine (Tyr450) 07-1643 1:1000

ATR 09-070 1:500

BRCA-1 clone BC70 05-842 1:500

CAF1 p60, clone SS-53, 1-124 04-1523 1 µg/mL

Catenin, b 06-734 1:200

c-Jun, clone 6E4.4 05-1076 5 µg/mL

CREB (bZIP transcription factor) 06-863 1:200

CyPA (Cyclophilin A) 07-313 1:1000

Endophilin B1 AB10555 1:500-1:1000

FUBP3 07-742 2 µg/mL

G9a (BAT8) 09-071 1 µg/mL

GAPDH MAB374 1:8000

HDAC11 09-827 0.5 µg/mL

Hexim 1 07-955 4 µg/mL

HLX1 09-084 2 µg/mL

IGF2 mRNA-binding protein 2 07-103 4 µg/mL

IGF2 mRNA-binding protein 3 07-104 2 µg/mL

IKKa 07-1007 1 µg/mL

IKKb 07-1008 2-10 µg/mL

IRS1, clone 58-10C-31 05-784R 0.1 µg/mL

JunD 07-1334 1:2000

LSM14A ABE37 1 µg/mL

MAP Kinase 1/2 (Erk-1/2) 06-182 1:500

MYPT1, phosphothreonine (Thr696) ABS45 0.1 µg/mL

NES, Nestin AB5922 1:1000

NEFL, 70 kDa clone DA2 MAB1615 1:200

P38/SAP-K2 05-454 1:600

P 53 AB565 1:1000

PLCg-1, phosphotyrosine (Tyr783) 07-2134 1:1000

PLK1, phosphoserine (Ser137) 07-1348 0.5 µg/mL

Post-immunodetection chromogenic staining of blots When weak signals were obtained in lanes 5 & 6, blots were stained with Coomassie blue following either chemiluminescence (left blots) or fluorescence (right blots) detection to verify equal loading and protein transfer of all lanes.

Lane 1: Molecular weight marker Lane 2: A431 Pervanadate (PVD) stimulated Lane 3: A431 control non stimulated

(fresh samples)Lane 4: A431 EGFR stimulated

(fresh samples)Lane 5: A431 control non stimulated

(old samples)Lane 6: A431 EGFR stimulated

(old samples)

1 2 3 4 5 6 1 2 3 4 5 6

Chemiluminescence detection

Blot stained after detection

1 2 3 4 5 6 1 2 3 4 5 6

Fluorescence detection

Blot stained after detection

ORdERINg INfORMATIONDescription Detection Method Qty/Pk Catalogue No.

Bløk-CH Reagent Chemiluminescence detection 500 mL/bottle WBAVDCH01

Bløk-FL Reagent Fluorescence detection 500 mL/bottle WBAVDFL01

Bløk-PO Reagent Phosphoprotein detection 500 mL/bottle WBAVDP001

BlockingAntibody Incubation

Bløk-PO reagent preserves the phosphorylation state of the protein

Chemiluminescence detection of pERK in EGF-stimulated A431 lysate (10-2.5 µg/lane,12-110, Millipore) using anti-pERK antibody (1:200, 05-797R, Millipore). Bands were detected using Luminata Forte Western HRP substrate (WBLUF0500, Millipore). pERK phosphorylated doublet indicated by arrow. NFDM - Non-fat dry milk

Competitor L Competitor P NFDM Bløk – PO

SNAP i.d. PublicationsMihrshahi R, Barclay AN, Brown MH. J Immunol. 2009 Oct 15; 183(8):4879-86.

Fujimori K, Ueno T, et al. J Biol Chem. 2010 Mar 19; 285(12):8880-6.

Sakane A, Honda K, Sasaki T. Mol Cell Biol. 2010 Feb; 30(4):1077-87.

Many more to be found on www.millipore.com/SnapPub.

Validated for the SNAP i.d. system

For a complete listing, visit the SNAP i.d. Antibody Optimization Reference Guide at www.millipore.com/SNAPab.

10 11

The new Luminata Western HRP Substrates

are a family of premixed, ready-to-use

chemiluminescent reagents for the detection

of HRP-based Westerns. Pour directly onto

the Western membrane without worrying

about pipetting error. The Luminata Classico,

Crescendo and Forte substrates cover a broad

range of sensitivities for all detection needs.

Luminata Western HRP SubstratesPremixed for convenience. Formulated for optimal results.

KEy fEATuRES• Conveniently premixed to avoid

cross contamination of reagents

• Broad range of sensitivities to

cover all detection needs

• Consistent results with less

pipetting error

• Stable at 4 °C or room temperature

• Most sensitive detection reagents

• Compatible with PVDF and

nitrocellulose membranes

Luminata substrates provide better protein detection.Dilution series of stimulated A431 lysates (10-0.6 µg) were resolved by SDS-PAGE then transferred on to Immobilon P membrane. Blots were blocked with Bløk-CH reagent and probed with either anti-PP2, anti-STAT1 (05-987, Millipore), or anti-BRCA1 (05-842, Millipore) primary antibody diluted in Bløk-CH reagent. The appropriate secondary antibodies were added to each antibody. Each blot was visualized with the indicated HRP detection reagent. The limit of detection for each reagent is indicated in left column. Blots were processed using the SNAP i.d. protein detection system.

Luminata Classico Luminata Crescendo Luminata Forte

Benefit Premixed reagent for everyday detection needs

Premixed reagent for detection within the low picogram range

Premixed reagent for detection within the femtogram range

detection limit ~ 6 picograms ~1-3 picograms ~ 400 femtograms

Stock solution stability 1 year at 4 °C 1 year at 4 °C 1 year at room temperature

Related Products

Western Blot Enhancing Reagents Avoid repeating your Western blotting experiment. Enhance your signal with the ChemiLucent™ Plus kit or strip

and reprobe your blot with a different antibody using the ReBlot™ Plus or Blot Restore reagents.

Description Qty/Pk Catalogue No.

ChemiLucent Plus Western Blot Enhancing Kit 1 kit 2650

ReBlot Plus Mild Antibody Stripping Solution, 10X 50 mL 2502

ReBlot Plus Strong Antibody Stripping Solution, 10X 50 mL 2504

Blot Restore Membrane Rejuvenation Kit, 10X 1 kit 2520

ORdERINg INfORMATION

Description Qty/Pk Catalogue No.

Luminata Classico Western HRP Substrates 100 mL WBLUC0100

Luminata Classico Western HRP Substrates 500 mL WBLUC0500

Luminata Crescendo Western HRP Substrates 100 mL WBLUR0100

Luminata Crescendo Western HRP Substrates 500 mL WBLUR0500

Luminata Forte Western HRP Substrates 100 mL WBLUF0100

Luminata Forte Western HRP Substrates 500 mL WBLUF0500

Immobilon Western Chemiluminescent HRP Substrate 100 mL WBKLS0100

Immobilon Western Chemiluminescent HRP Substrate 500 mL WBKLS0500

Protein: PP2~ 6 picograms

Luminata Classico Competitor P Competitor G

Protein: STAT1~ 1-3 picograms

Luminata Crescendo Competitor P Competitor B

Luminata Forte Competitor P Competitor GProtein: BRCA1~ 400 femtograms

detection

Detection of GAPDH in 10 µg (Lane 1), 5 µg (Lane 2), 2.5 µg (Lane 3), and 1.2 µg (Lane 4) of A431 lysate. Blots were treated with the indicated detection reagent and exposed to x-ray film for 5 minutes. Luminata Forte substrate is equivalent to Immobilon Western Chemiluminescent HRP substrates.

Luminata Classico Luminata Crescendo Luminata Forte

Validated for the SNAP i.d. system

10 11

The new Luminata Western HRP Substrates

are a family of premixed, ready-to-use

chemiluminescent reagents for the detection

of HRP-based Westerns. Pour directly onto

the Western membrane without worrying

about pipetting error. The Luminata Classico,

Crescendo and Forte substrates cover a broad

range of sensitivities for all detection needs.

Luminata Western HRP SubstratesPremixed for convenience. Formulated for optimal results.

KEy fEATuRES• Conveniently premixed to avoid

cross contamination of reagents

• Broad range of sensitivities to

cover all detection needs

• Consistent results with less

pipetting error

• Stable at 4 °C or room temperature

• Most sensitive detection reagents

• Compatible with PVDF and

nitrocellulose membranes

Luminata substrates provide better protein detection.Dilution series of stimulated A431 lysates (10-0.6 µg) were resolved by SDS-PAGE then transferred on to Immobilon P membrane. Blots were blocked with Bløk-CH reagent and probed with either anti-PP2, anti-STAT1 (05-987, Millipore), or anti-BRCA1 (05-842, Millipore) primary antibody diluted in Bløk-CH reagent. The appropriate secondary antibodies were added to each antibody. Each blot was visualized with the indicated HRP detection reagent. The limit of detection for each reagent is indicated in left column. Blots were processed using the SNAP i.d. protein detection system.

Luminata Classico Luminata Crescendo Luminata Forte

Benefit Premixed reagent for everyday detection needs

Premixed reagent for detection within the low picogram range

Premixed reagent for detection within the femtogram range

detection limit ~ 6 picograms ~1-3 picograms ~ 400 femtograms

Stock solution stability 1 year at 4 °C 1 year at 4 °C 1 year at room temperature

Related Products

Western Blot Enhancing Reagents Avoid repeating your Western blotting experiment. Enhance your signal with the ChemiLucent™ Plus kit or strip

and reprobe your blot with a different antibody using the ReBlot™ Plus or Blot Restore reagents.

Description Qty/Pk Catalogue No.

ChemiLucent Plus Western Blot Enhancing Kit 1 kit 2650

ReBlot Plus Mild Antibody Stripping Solution, 10X 50 mL 2502

ReBlot Plus Strong Antibody Stripping Solution, 10X 50 mL 2504

Blot Restore Membrane Rejuvenation Kit, 10X 1 kit 2520

ORdERINg INfORMATION

Description Qty/Pk Catalogue No.

Luminata Classico Western HRP Substrates 100 mL WBLUC0100

Luminata Classico Western HRP Substrates 500 mL WBLUC0500

Luminata Crescendo Western HRP Substrates 100 mL WBLUR0100

Luminata Crescendo Western HRP Substrates 500 mL WBLUR0500

Luminata Forte Western HRP Substrates 100 mL WBLUF0100

Luminata Forte Western HRP Substrates 500 mL WBLUF0500

Immobilon Western Chemiluminescent HRP Substrate 100 mL WBKLS0100

Immobilon Western Chemiluminescent HRP Substrate 500 mL WBKLS0500

Protein: PP2~ 6 picograms

Luminata Classico Competitor P Competitor G

Protein: STAT1~ 1-3 picograms

Luminata Crescendo Competitor P Competitor B

Luminata Forte Competitor P Competitor GProtein: BRCA1~ 400 femtograms

detection

Detection of GAPDH in 10 µg (Lane 1), 5 µg (Lane 2), 2.5 µg (Lane 3), and 1.2 µg (Lane 4) of A431 lysate. Blots were treated with the indicated detection reagent and exposed to x-ray film for 5 minutes. Luminata Forte substrate is equivalent to Immobilon Western Chemiluminescent HRP substrates.

Luminata Classico Luminata Crescendo Luminata Forte

Validated for the SNAP i.d. system

Millipore, Advancing Life Science Together, SNAP i.d., Immobilon, Amicon, and Ultracel are registered trademarks of Millipore Corporation. ReNcell, PureProteome, Luminata, Bløk, ReBlot, ChemiLucent, and the M mark are trademarks of Millipore Corporation. Qdot is a trademark of Quantum Dot Corporation. Kodak is a registered trademark of Eastman Kodak Company. Lit. No. PB1033EN00 Printed in the USA 03/10 LS-SBU-10-02831 ©2010 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

Immobilon Transfer MembranesDescription Size Qty/Pk Catalogue No.

Immobilon P: PVDF 0.45 µm 7 x 8.4 cm 50/pk IPVH07850

26.5 cm x 3.75 m 1 roll IPVH00010

Immobilon PSQ: PVDF 0.2 µm 7 x 8.4 cm 50/pk ISEQ07850

26.5 cm x 3.75 m 1 roll ISEQ00010

Immobilon FL: PVDF 0.45 µm 7 x 8.4 cm 10/pk IPFL07810

26.5 cm x 3.75 m 1 roll IPFL00010

SNAP i.d. System

Description Components Qty/Pk Catalogue No.

SNAP i.d. Protein Detection System WBAVDBASE

SNAP i.d Consumables and Accessories Single Blot Holder 30/pk WBAVDBH01

Double Blot Holder 30/pk WBAVDBH02

Triple Blot Holder 20/pk WBAVDBH03

Antibody Collection Tray 20/pk WBAVDABTR

SNAP i.d. Blot Roller 1/pk WBAVDR0LL

Bløk Noise-Canceling ReagentsDescription Detection Method Qty/Pk Catalogue No.

Bløk-CH Reagent Chemiluminescence Detection 500 mL/bottle WBAVDCH01

Bløk-FL Reagent Fluorescence Detection 500 mL/bottle WBAVDFL01

Bløk-PO Reagent Phosphoprotein Detection 500 mL/bottle WBAVDP001

Luminata Western HRP SubstratesDescription Qty/Pk Catalogue No.

Luminata Classico Western HRP Substrate

500 mL WBLUC0500

Luminata Crescendo Western HRP Substrate

500 mL WBLUR0500

Luminata Forte Western HRP Substrate

500 mL WBLUF0500

Western Blotting Enhancing ReagentsDescription Qty/Pk Catalogue No.

ChemiLucent Plus Western Blot Enhancing Kit

1 kit 2650

ReBlot Plus Mild Antibody Stripping Solution, 10x

50 mL 2502

ReBlot Plus Strong Antibody Stripping Solution, 10x

50 mL 2504

ORdERINg INfORMATION

Western Blotting ToolsPublication Quality Westerns in Minuteswith Millipore’s Pre-optimized Products

TO PLACE AN ORdER In the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)

In Europe, please call Customer Service:

France: 0825.045.645 • Spain: 901.516.645 Option 1 • Germany: 01805.045.645 • Italy: 848.845.645 • UK: 0870.900.46.45

For other countries across Europe and the world, please visit www.millipore.com/offices.

For Technical Service, please visit www.millipore.com/techservice.

www.millipore.com/WBtools