Hopefully someone can shed a little light on this topic for me.

I am interested in covalently linking an antibody to protein-A sepharose immobilized on beads. There is a protocol for this, which uses dimethyl pimelimidate hydrochloride to induce covalent crosslinking between the protein-A and Ig. However, once the protein-A is covalently linked to the Ig, is it then possible to repeat the same protocol to covalently link a second antibody to the antibody already attached to the protein A? So in effect, I am trying to link protein-A to a secondary antibody, which I then want to link to a primary antibody for use in IPs. I need them to be covalently linked so they don't elute with my protein of interest in the last stages of my IP.

And no I cannot just link the primary antibody directly to the protein-A.

Anyone?

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hola...

first of all you got to know wether the cross linking reagent you use is homo- or heterofunctional and to what groups (thiols or primery amines) it´s reactive...if possible, you should check then your secondary antibody (coupled first to Prot-A) for those groups in its epitope binding site...if your secondary antibody contains such groups it might be possible that the secondary Ab loses its affinty to your primary Ab...

if so, let your primery Ab bind to your sec. Ab and add the prot-a afterwards. Then you should be able to cross link primery Ab + sec. Ab + prot-A in one reaction step.

viel glück dabei...good luck...

moses

Why can't you crosslink directly your primary antibody? Have you checked wether it has affinity for Prot G?

In case you want to couple primary and sec antibodies, I think that Moses is right: it's a better idea to let bind both antibodies first, and then add them to the prot A for the crosslinking step.