To avoid this problem, I was thinking different ratio of antibody and protein A might help..., but is too saturated antibody in protein A resin? or less antibody in protein A resin? can help remain binding affinity of antibody after cross link?
If your antibody is losing affinity after chemical crosslinking to protein A, it is probably because the crosslinker is reacting with the antigen binding sites. Possible solutions are (1) not to crosslink the antibody to the protein A resin, (2) to use a different crosslinking chemistry, (3) to protect the antigen binding sites with antigen during the crosslinking step, or (4) to minimize the crosslinker concentration.
Hi Shen,
I think using more antibody will actually help you to coat the protein A resign well.
Basically you will be blocking all of the active sites with your antibody.
So if you want to be sure about your coating its better to use more antibody.
good luck !!!
If your antibody is losing affinity after chemical crosslinking to protein A, it is probably because the crosslinker is reacting with the antigen binding sites. Possible solutions are (1) not to crosslink the antibody to the protein A resin, (2) to use a different crosslinking chemistry, (3) to protect the antigen binding sites with antigen during the crosslinking step, or (4) to minimize the crosslinker concentration.
Hello Bj Shen@
I think that there is no problem with the interaction , because the primary binding site for protein A is on the Fc region, between the CH2 and CH3 domains , so i guess that you have to controle the pH of the buffer or the suspenssion that you have added to protect and to stabilize your complex (proteinA -Fc Ig).
You have also to verify its temprature , more it's low , the time of complexation is long.
Good luck.
Have you carried out immuno-chemical cross linking or chemical cross linking of antibody with protein-A resin? If immuno-chemical cross linking has been done than antigen binding site will remain free because protein-A binds at Fc region of the antibody. If this complex you have preserved in simple buffer than in due course of time antibody will lose its binding property. Therefore you have to protect antibody activity by adding additives.
If chemical cross linking of antibody with protein-A resin has been done than there is a possibility that antigen site got cross linked with protein thereby it is not binding to antigen. Secondly the type and quantity of solvent and chemical cross-linker used may have a affect at binding site of antibody. That you have to ascertain at your end.
If you require further information please get back to me.
With best wishes.
Certainly I would test different ratios of Ab:resin, as too high a binding and/or crosslinking could cause loss of affinity.
I would also consider using pre-made activated resins such as NHS-activated Sepharose or CNBr. Certainly you do not have the directed binding with protein A but you do not introduce the variability that chemical crosslinking may bring post binding.
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