How to elute antigen during immunoprecipitation experiment with out primary anti (View forum version)



chimera

Posted 13 December 2012 - 11:47 PM

My protein (50 kDa), which I am trying to immunoprecipitate matches with the heavy chain of primary antibody. So I want to elute only my antigen without disturbing the primary antibody, so that I can detect it on the western. How can I do that? Please help me. Lots of thanks in advance.

bob1

Posted 14 December 2012 - 12:32 AM

You may be able to elute using 0.1 M glycine pH 2.5. You can also buy protein G-HRP/alk phos conjugates for getting around this problem - after you have eluted and run on a denaturing gel, the denatured antibody chains will no longer be recognised by the protein-G conjugate, whereas your native intact detecting antibody will be bound by the protein G.