Antibody & Protein Labeling Kits · immunohistochemistry(IHC) applications requiring several hours...

Jean-Michel CocchiInvitrogen Cellular Analysis

[email protected]

Antibody & Protein Labeling Kits

| Life Technologies Proprietary & Confidential | 2

Direct VS. Secondary Detection

Direct-Labeled Primary Antibody

• Low background

• Covalent attachment of label via amine-reactive dyes to lysines

• Labeling could affect antigen recognition site

• Fluorescent signal similar to Zenon®-Labeled Antibody

Indirect-Labeled Secondary Antibody or Streptavidin

• Higher Background

• Brighter Signal

Zenon®-Labeled Antibody

• Low background

• Non-covalent attachment of Zenon® fragment to Fc portion

• Labeling will NOT affect antigen recognition site

• Fluorescent signal similar to Direct-labeled Primary antibody

• For use with “higher” abundance targets

• To reduce background due to non-specific secondary detection

• For use with multiplexed analyses with same-species antibodies


How to Choose an Antibody Labeling Kit....1) Do I need a directly labeled conjugate?

2) What

label?

For an interactive version visit:

www.invitrogen.com/spectraviewer


How to Choose An Antibody Labeling Kit....3) How much antibody do I want or need to label?

1-20 µµµµg IgG 20-100 µµµµg IgG 100 µµµµg IgG 1 mg IgG

Zenon® IgG Labeling Kits

APEX Antibody Labeling Kits

Microscale protein labeling kits

Monoclonal antibody labeling kits

“Original” Protein labeling Kits


How to Choose An Antibody Labeling Kit....4) Should the label be covalently or non-covalently

attached to the antibody?Covalent attachment Non-Covalent label attachment

These conjugates are stable for months to even years…

Can be used with any antibody application

Products:

• APEX Antibody Labeling kits

• Microscale protein labeling kits

• Monoclonal antibody labeling kits

• Protein labeling kits

Conjugates are stable for hours not days…

Recommended for use with applications requiring short incubations

NOT recommended for immunohistochemistry (IHC) applications requiring several hours to overnight antibody incubations

Product:

• Zenon® Antibody Labeling Kits

Label directly & covalently attached to the IgG antibody

Label non-covalently attached to the IgG antibody using directly labeled anti-FC fab fragments


How to Choose an Antibody Labeling Kit5) Does the antibody contain stabilizing proteins?Typically:

• The stabilizing is bovine serum albumin (BSA)

• This is used to stabilize small amounts of packaged IgG antibodies

• Whether the IgG contains BSA will either be listed on the vial or in the product data sheet

Why does this matter?

If labeling with a microscale, monoclonal & protein labeling kit, these kits utilize amine-reactive dyes that will react with the stabilizing proteins in addition to the IgG antibody. If using a kit with amine-reactive dyes and the stabilizing proteins are not removed, the antibody will be underlabeled and produce a very weak signal.

Zenon® and APEX antibody labeling kits are completely compatible with stabilizing proteins.


Zenon® vs non-Zenon® Antibody Labeling KitsZenon® Antibody Labeling

APEX, Microscale, Monoclonal Antibody & Protein Labeling Kits

• Fastest way to label IgG (10 minutes!!)

• Very, very easy-to-use

• Perfect for small quantities of IgG (<20 µµµµg)

• Great way to attach, Alexa Fluor® dyes R-PE, R-PE-tandems (and other fluorescent proteins or enzymes)

• Fast (1.5-2 hours, only 15-30 minutes of actual hands-on time)

• Very easy-to-use

• Perfect for larger quantities of IgG (>20 µµµµg – 1 mg)

• Great way to covalently attach Alexa Fluor® dyes & biotin


~ 1 µg primary antibody

Add Zenon®

reagent at desired molar ratio

Incubate 5 minutes

Block with non-specific IgG

& use directly

How does Zenon Labeling work?


Kinetically trapped complex formation: key to making the system work.

Fast complex formation Slow complex dissociation

+ Alexa Fluor 488 ZenonCapture by BSA-biotin in microtiteer plate

Mouse anti-biotin IgG1

Control IgG1

Blocked mouse anti-biotin IgG1

+ Alexa Fluor 488 ZenonCapture by BSA-biotin in microtiteer plate

Zenon labeling technology- Stability


Use in imaging…

• anti-nuclear mouse mAb + Alexa Fluor 350 Zenon labeling reagent• F-actin + Alexa Fluor 488 phalloidin• anti-tubulin mouse mAb + Alexa Fluor 568 Zenon labeling reagent


Use of Zenon® Labeling Technology in flow cytometry

• Human peripheral blood lymphocytes were stained with the following three antibodies: an anti-CD3 mouse IgG1 antibody (A21330) prelabeled with the Zenon® Alexa Fluor® 647 Mouse IgG1 Labeling Kit (Z25008), an anti-CD4 mouse IgG1 antibody (A21334) prelabeled with the Zenon® R-Phycoerythrin Mouse IgG1 Labeling Kit (Z25055) and an anti-CD8 mouse IgG2a antibody (A21338) prelabeled with the Zenon® Alexa Fluor® 488 Mouse IgG2a Labeling Kit (Z25102). Panels A and B show that cells can be separated by plotting the orange-fluorescent versus green-fluorescent signal or red-fluorescent versus orange-fluorescent signal, respectively, demonstrating that Zenon® labeling technology does not transfer to other antibodies in the same sample. The samples were analyzed on a Coulter Elite flow cytometer using 488 nm excitation for R-phycoerythrin and the Alexa Fluor® 488 dye, and 633 nm excitation for the Alexa Fluor® 647 dye.


APEX Antibody Labeling Kits• Convenient: APEX Kits label small amounts of antibody, 10-20 µµµµg

• Covalent attachment of label: Once attached, label will not come off

• Compatible: Stabilizing proteins (i.e., BSA) or amine-containin g buffers (i.e., TRIS) will not interfere with labeling

Customer requirements:

• 10-20 µg IgG antibody

• Standard pipette (for 200 µL volume)

• ~15 minutes “hard labor” (2.5 hours total)

With pipette, apply to APEX labeling tip

• Apply IgG antibody

• Fluorescent label

• Wash buffers

APEX antibody labeling tip

With pipette, elute

• Wash buffers

• Labeled IgGMultiple steps: Customer will need to either apply or elute


APEX or Zenon ?

APEX Labeled Antibody

• Lowest background

• Labeling could affect antigen recognition site

• Fluorescent signal similar to Zenon®-Labeled Antibody

• 10-20 µµµµg IgG per labeling

• Attach simple, organic dyes (Alexa Fluor®, Oregon Green® dyes for use in flow cytometry or microscopy or even as haptens with anti-Alexa Fluor or Anti-Oregon Green antibodies)

Indirect-Labeled Secondary Antibody or Streptavidin

• Higher Background

• Brightest Signal

Zenon®-Labeled Antibody

• Low background

• Fc-attachment of label. Labeling will NOT affect antigen recognition site

• 1-20 µµµµg IgG per labeling

• Isotype specific labeling (IgG2a or IgG2b

• Attach

• Simple organic dyes (for use in flow cytometry or microscopy)

• Phycobiliproteins (R-PE, APC for flow cytometry)

• Tandems (Alexa Fluor-APC for flow cytometry)

Antibody

Antigen

Label


Antibody & Protein Labeling Kits OverviewAPEX Antibody Labeling Kits

Microscale Protein Labeling Kits

Monoclonal Antibody Labeling Kits

Protein Labeling Kits

*Requires optimized primary antibodies to target or short incubations (<1 hour)

Flow Cytometry

Microscopy

• ICC

• IHC

Applications

Zenon® Antibody Labeling Kits

SAIVI™ Protein / Antibody Labeling Kits

QDot® Nanocrystal Secondary Antibody Labeling Kits

Applications

Small Animal in-vivoImaging (SAIVI™) Applications

Flow Cytometry

Microscopy

• ICC

• IHC*

Applications*

Flow Cytometry *

Microscopy *

• ICC

• IHC

Western blots

SAIVI™ Applications

Applications

* Verify ex/em resources

Available Labels

Alexa Fluor® Dye Series

Biotin and DSB™-X biotin

Oregon Green® Dyes

Pacific™ Dyes

Standard Dyes (fluorescein)

Available Labels

Near IR Alexa Fluor® Dyes

• Alexa Fluor® 647, 680 & 750

Available Labels

Alexa Fluor® Dye Series

Alexa Fluor® tandems

Phycobiliproteins (R-PE, APC)

Biotin and DSB™-X biotin

Oregon Green® Dyes

Pacific™ Dyes

Standard Dyes (fluorescein)

Available Labels

QDot® Nanocrystals

• 525, 565, 585, 605, 655, 705, 800

• Covalent label attachment

• DOL optimized for brightest signal


• DOL optimized for SAIVI™Applications

• DOL control reagent

• 5 minute antibody labeling

• Non-covalent label attachment

• Organic dyes & R-PE


Jean-Michel COCCHI – TSSAnalyse de la [email protected]

Fluorescence amplification with Tyramide kits


TSA™ can provide high resolution and high S/N

Tyramide Signal Amplification (TSA™)


Why Use TSA?

• Generates multiple copies of Alexa Fluor dyes at ta rget site

• Gives high-resolution signal amplification

• When normal amplification methods fail− Low abundance targets

Calbindin Revealed


StreptavidinAlexa Fluor

546

Alexa Fluor 546 TSA

Alexa Fluor 546 Dye FISH Signal: Streptavidin vs TSA

0

500

1000

1500

RFU

AF546-SA AF546-TSA

10-fold increase in sensitivity w/ TSA™

Chromosome FISH: Labeled Streptavidin vs TSA™


Labelling examples on Zebrafish sample

A zebrafish retina cryosection labeled with the mouse monoclonal antibody FRet 43 and detected using TSA Kit #9 with the HRP conjugate of goatanti–mouse IgG antibody and Alexa Fluor® 488 tyramide.


Labelling examples on Zebrafish sample

Zebrafish larva reticulospinalneurons. AlexaFluor® 488 tyramide .


Why Use TSA?• Increased sensitivity

− ~100-fold compared to avidin–biotinylated enzyme complex (ABC)

• Generates multiple copies of AlexaFluor dyes at target site

• Gives high-resolution signal amplification

• When normal amplification methods fail

− Low abundance targets

3 color sequential TSA™


Protecting the fluorescence signal-Antifade Reagents

Prolong® Gold

•Optimized formulation-Superior antifade with little or no impact on initial fluorescence intensity

•Ready to use - dropper bottle

•Cures to a firm gel – no more nail polish

NEW: SlowFade® Gold - antifade mounting media which doesn’t polymerize

Antibody & Protein Labeling Kits · immunohistochemistry(IHC) applications requiring several hours...

Documents

Transcript of Antibody & Protein Labeling Kits · immunohistochemistry(IHC) applications requiring several hours...