Hi,
I have performed several conjugations with different antibodies (Human IgG, Mouse CRP) to 200 nm carboxylated beads using two-step EDAC/Sulfo-NHS method.
The protocol, method and materials I am using have been enough to conjugate the human IgG and Mouse CRP antibodies to the beads with about 65% coupling efficiency.
I am testing a new sheep monoclonal antibody and I cannot get it to conjugate AT ALL to the same 200 nm beads with the same protocol.
Any advice?
Hi Sarah Ashby . You can skip the sulfo-NHS intermediate step entirely and just incubate the carboxyl beads with EDC and antibody in MES buffer pH 5.5.
Dear Sarah,
check the buffer your antibody came in and if there are any other components like albumin. Also purity (e.g. whole serum, ascites or total IgG fraction) might be issue.
Carbodiimide chemistry does not work when there are amino groups in the buffer, like e.g. when you have Tris. With other proteins present, you'll get a crosslinked mess.
Steingrimur Stefansson, when you do the one pot reaction, wouldn't you get a lot of crosslinking of antibodies, which will yield a different coupling pattern to the beads, compared to activating them first as NHS esters and then removing excess reagent?
Hi Wolfgang Schechinger . Some protein X-linking is unavoidable using EDC straight, but the efficiency is so much higher than going through the NHS intermediate. The reaction is easier to control if you are dealing with one COOH group to couple with proteins.
You can minimize protein X-linking by doing sequential addition (e.g. EDC + COOH, 5 min incubation, then add protein). You can also adjust the concentrations (e.g. COOH + 5X molar XS EDC, incubate, then add protein.)
For COOH resins, you can incubate with EDC, centrifuge, remove the sup. that has XS EDC and replace it with the protein solution.
Antibody is in a PBS buffer with .1% sodium azide. I find it very strange that other antibodies bind so readily and this antibody gives less than 5% efficiency.