immunoprecipitation troubleshooting - (Oct/28/2012 )
I have the problem of immunoprecipitation. Please help me solve this problem.
I overexpressed the HA-tag expression vector in cells，and i can detect the exogenous HA tag protein by using western blotting.
But when I perform immunoprecipitation, I used HA tag antibody to pull down the protein complex of my HA-tag protein. I cant detect the HA-tag protein in the pulldown protein complex by using western blotting. My HA-tag protein has many zinc finger, i think whether the HA tag is embed in the protein structure, so HA tag antibody cant pull down this protein.
Please help me disolve this problem.
Thanks very much.
it does sound like your HA tag is inaccessible to the antibody. You could try different detergents to change conformation and complexes slightly. You may also try inserting the HA tag in another region of the protein, maybe the other end of the polypeptide chain, or even insert it within the coding sequence.