immunoprecipitation and western blotting assay by the same antibody - (Jun/04/2006 )

hi,
I have a question about the immunoprecipitation and the followed western blot assay by the same antibody. The background is always high. I have not find another antibody against my target protein. How can I do to lower the background?
Thanks for your help.

I will try this method, thank you.

what do you mean by background?
is it the denatured antibody used for IP?
then you could use either a biotinylated antibody for western-blot, or use protein A -HRP or protein G-HRP instead of secondary antibody .

You need to chemically tag the antibody so you do not need to use a secondary to detect the antigens. This is a common problem that will prevent you from seeing your immunoprecipitated antigens because of the large amount of heavy and light chains present on the gel of your immunoprecipitates which bind your secondary antibody in the Western analysis. A key to success in this regard is having a highly, purified functional antibody to both IP and use for chemical tagging (eg., biotinylation). We have developed techniques for producing such highly affinity purified probes used in just such applications. Take a look at our TrpC antibody blog at http://trpc.blogware.com/blog. We have used these particular doubly affinity purified synthetic peptide antibodies to IP the TrpC calcium channel subunits from tissues and detect them on Westerns using their biotinylated counterparts. We have also obtained sufficient amounts of the antigens from immunoprecipitation reactions to mass spec and confirm their identities. The key to successful isolation and identification of low abundance antigens is highly purified, functional antibodies. Contact me if you need help in this regard (416-673-8155 or toll free 877-873-8155)