Affinity purifed Polyclonal, better than monoclonal for IP? - (Apr/14/2010 )
I am using the Direct IP kit from Pierce for immunoprecipitation, with 2ug of affinity purified polyclonal antibody coupled to the amino resin.
This antibody is very expensive and we were wondering if it should be replaced by a cheaper monoclonal antibody.
The kit protocol simply explains that the antibody must be affinity-purified and be free of serum proteins.
It is equivalent a monoclonal antibody to a highly purified polyclonal?
EGF on Apr 14 2010, 07:09 AM said:
no. polyclonal will be active against multiple epitopes, monoclonal against one.
Any antibody will either work or not work for immunoprecipitation, so you just have to test it. It looks like your kit requires the antibody to be purified without any BSA or primary amines. Cheap monoclonals will be supplied as ascites fluid, with some glycerol, azide, tris etc. Many of these things would interfere with your linkage. You probably just want to special order a good antibody in PBS. There are companies that will provide custom formulated antibodies for these applications. Alternatively, you could just use standard protein A or protein G agarose, pre-bind the antibody to the beads, wash away the "extra stuff" and cross-link your antibody to the protein A/G beads using a cross-linking protocol (DMP maybe?).