Antibody conjugation to SAMs - advice?

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PHDgirl
PHDgirl's picture
Antibody conjugation to SAMs - advice?

I've been trying to conjugate anti-biotin to SAMs of 3-mercaptopropanoic acid (MPA) (deposited on silver films), by first activating the COOH group using NHS (n-hydroxy succinimide) and EDC (ethyl dimethylaminopropyl carbodiimide) in buffer solution, and then rinsing in more buffer and drop coating the slides with the antibody solution (I would like to immerse the slides, but the antibody is too expensive!) for 90 mins at 4 degrees, then rinsing with buffer. I then do spectroscopy on the slides to see if the antibodies have conjugated - I'm not getting great results though!

I've been using buffer at pH 5.5 throughout (with KH2PO4, K2HPO4 and NaCl).

Am I doing anything obviously wrong/ do you have any tips/advice? Anything you can tell me will help, since I'm a physicist by trade and all this biochemistry is very new to me!

Tony Rook
Tony Rook's picture
PHDgirl:

PHDgirl:

I would recommend you trying to get your hands on the following book:

Hermanson, GT (ed). Bioconjugate Techniques. Academic Press, San Diego, 1996.

A couple chapters (and their protocol titles) might be of interest to you:

Chapter 10 - Antibody Modification and Conjugation

NHS Ester-Melemimide-Mediated Conjugation
Activation of Enzymes with NHS Ester-Maleimide
Cross-linkers

Conjugation with Reduced Antibodies

Conjugation with 2-Iminothiolane-Modified Antibodies

Conjugation with SATA-Modified Antibodies

Glutaraldehyde-Mediated Conjugation

One-Step Glutaraldehyde Protocol

Two-Step Glutaraldehyde Protocol

Reductive-Amination-Mediated Conjugation

Activation of Enzymes with Sodium Periodate

Activation of Antibodies with Sodium Periodate

Conjugation of Periodonate-Oxidized HRP to Antibodies
by Reductive Amination

Conjugation of Periodate-Oxidized Antibodies with Amine
or Hydrazide Derivatives

Conjugation Using Antibody Fragments
Preparation of F(ab')2 Fragments Using Pepsin

Preparation of Fab Fragments Using Papain

Removal of Unconjugated Enzyme from Antibody-Enzyme
Conjugates
Immunoaffinity Chromatography

Nicke-Chelate Affinity Chromatography

Chapter 13 - Avid-Biotin Systems

NHS Ester-Maleimide-Mediated Conjugation Protocols
Protocol for the conjugation of SMCC-Activated Avidin or
Streptavidin with Thiolated Enzyme

Periodate Oxidation/Reductive Amination Conjugate Protocols
Protocol for the Conjugation of Avidin with Ferritin Using
Reductive Amination

Protocol for the Preparation of Avidin-HRP or
Streptavidin-HRP by Reductive Amination

Glutaraldehyde Conjugation Protocol
Protocol for the One-Step Glutaraldehyde Conjugation of
Ferritin to Avidin or Streptavidin

Protocol for the Two-Step Glutaraldehyde Conjugation of
Enzymes to Avidin or Streptavidin

Hope this helps!

bwbrian
bwbrian's picture
I've activated beads with EDC

I've activated beads with EDC and Sulfo-NHS, a couple tips from my experience. Make sure you don't have any amine-containing (or other nucleophile) components in your buffers. The other thing is that you are forming NHS-esters wihen you activate with EDC and Sulfo-NHS. These are really reactive ... they even react with water, so keep the pH down and add your antibody asap (before the active esters hydrolyze). If you do this, the reaction should be really smooth and easy.