Antibody & Protein Labeling Kits. Jean-Michel Cocchi Invitrogen Cellular Analysis

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1 Antibody & Protein Labeling Kits Jean-Michel Cocchi Invitrogen Cellular Analysis

2 Direct VS. Secondary Detection For use with higher abundance targets To reduce background due to non-specific secondary detection For use with multiplexed analyses with same-species antibodies 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 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 Indirect-Labeled Secondary Antibody or Streptavidin Higher Background Brighter Signal 2

3 How to Choose an Antibody Labeling Kit... 1) Do I need a directly labeled conjugate? 2) What label? For an interactive version visit: 3

4 How to Choose An Antibody Labeling Kit... 3) How much antibody do I want or need to label? 1-20 µg IgG µg IgG 100 µg IgG 1 mg IgG Zenon IgG Labeling Kits Microscale protein labeling kits Monoclonal antibody labeling kits Original Protein labeling Kits APEX Antibody Labeling Kits 4

5 How to Choose An Antibody Labeling Kit... 4) Should the label be covalently or non-covalently attached Covalent to the attachment antibody? 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 5

6 How to Choose an Antibody Labeling Kit 5) 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. 6

7 Zenon vs non-zenon Antibody Labeling Kits Zenon Antibody Labeling Fastest way to label IgG (10 minutes!!) Very, very easy-to-use Perfect for small quantities of IgG (<20 µg) Great way to attach, Alexa Fluor dyes R-PE, R-PE-tandems (and other fluorescent proteins or enzymes) APEX, Microscale, Monoclonal Antibody & Protein Labeling Kits Fast (1.5-2 hours, only minutes of actual hands-on time) Very easy-to-use Perfect for larger quantities of IgG (>20 µg 1 mg) Great way to covalently attach Alexa Fluor dyes & biotin 7

8 How does Zenon Labeling work? ~ 1 µg primary antibody Add Zenon reagent at desired molar ratio Incubate 5 minutes Block with nonspecific IgG & use directly 8

9 Zenon labeling technology- Stability Kinetically trapped complex formation: key to making the system work. Fast complex formation Slow complex dissociation Mouse anti-biotin IgG 1 Blocked mouse anti-biotin IgG 1 + Alexa Fluor 488 Zenon Capture by BSA-biotin in microtiteer plate + Alexa Fluor 488 Zenon Capture by BSA-biotin in microtiteer plate Control IgG 1 9

10 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 10

11 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. 11

12 APEX Antibody Labeling Kits Convenient: APEX Kits label small amounts of antibody, µg Covalent attachment of label: Once attached, label will not come off Compatible: Stabilizing proteins (i.e., BSA) or amine-containing buffers (i.e., TRIS) will not interfere with labeling Customer requirements: 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 Multiple steps: Customer will need to either apply or elute With pipette, elute Wash buffers Labeled IgG 12 APEX antibody labeling tip

13 APEX or Zenon? Antibody Label Antigen APEX Labeled Antibody Lowest background Labeling could affect antigen recognition site Fluorescent signal similar to Zenon - Labeled Antibody µg 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) Zenon -Labeled Antibody Low background Fc-attachment of label. Labeling will NOT affect antigen recognition site 1-20 µg IgG per labeling Isotype specific labeling (IgG 2a or IgG 2b 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) Indirect-Labeled Secondary Antibody or Streptavidin Higher Background Brightest Signal 13

14 Antibody & Protein Labeling Kits Overview APEX Antibody Labeling Kits Microscale Protein Labeling Kits Monoclonal Antibody Labeling Kits Protein Labeling Kits SAIVI Protein / Antibody Labeling Kits Zenon Antibody Labeling Kits QDot Nanocrystal Secondary Antibody Labeling Kits Covalent label attachment DOL optimized for brightest signal Covalent label attachment DOL optimized for SAIVI Applications DOL control reagent 5 minute antibody labeling Non-covalent label attachment Organic dyes & R-PE Covalent label attachment Applications Applications Applications* Applications Flow Cytometry Microscopy ICC IHC Available Labels Small Animal in-vivo Imaging (SAIVI ) Applications Available Labels Near IR Alexa Fluor Dyes Alexa Fluor 647, 680 & 750 Flow Cytometry Microscopy ICC IHC* Available Labels Alexa Fluor Dye Series Flow Cytometry * Microscopy * ICC IHC Western blots SAIVI Applications Alexa Fluor Dye Series Biotin and DSB -X biotin Oregon Green Dyes Pacific Dyes Standard Dyes (fluorescein) 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, *Requires optimized primary antibodies to target or short incubations (<1 hour) * Verify ex/em resources

15 Jean-Michel COCCHI TSS Analyse de la cellule Fluorescence amplification with Tyramide kits

16 Tyramide Signal Amplification (TSA ) 16 TSA can provide high resolution and high S/N

17 Why Use TSA? Calbindin Revealed Generates multiple copies of Alexa Fluor dyes at target site Gives high-resolution signal amplification When normal amplification methods fail Low abundance targets 17

18 Chromosome FISH: Labeled Streptavidin vs TSA Streptavidin Alexa Fluor 546 Alexa Fluor 546 TSA Alexa Fluor 546 Dye FISH Signal: Streptavidin vs TSA 1500 RFU AF546-SA AF546-TSA fold increase in sensitivity w/ TSA

19 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 goat anti mouse IgG antibody and Alexa Fluor 488 tyramide. 19

20 Labelling examples on Zebrafish sample Zebrafish larva reticulospinal neurons. Alexa Fluor 488 tyramide. 20

21 Why Use TSA? Increased sensitivity ~100-fold compared to avidin biotinylated enzyme complex (ABC) Generates multiple copies of Alexa Fluor dyes at target site 3 color sequential TSA Gives high-resolution signal amplification When normal amplification methods fail Low abundance targets 21

22 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 22 NEW: SlowFade Gold - antifade mounting media which doesn t polymerize

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