phosphospecific antibody - how to get phosphospecific antibody (Dec/10/2004 )
I have questions about generation of phosphospecific antibody. I am studying a protein, which I have already shown, can be phosphorilated. And I want to prepare polyclonal Ab specific for phosphorilated form of the protein. I can express my protein in E. coli and phosphorilate it in vitro. What is better way to get a phosphospecific antibody: develop antibody against phosphopeptide or develop antibody against phosphorilated forms of the protein, and than deplete against non-phosphorilated forms? I know that company normally recommend use a phosphopeptides, but can be second way better?
Many thanks in advance!
It's not easy to make phosphospecific antibodies, and honestly, I would recommend getting a company to do it for you, and thus guarantee the quality and specificity. But otherwise, yes, you have to start off with a phosphopeptide, as using phosphorylated protein will most likely produce antibodies to other sites as well as the phospho-group.
Alternatively, you could test phosphotyrosine, -serine, and -threonine antibodies which are available. one of them might work with your protein.
I also suggest that you ask a company to do it. I had a good experience about phospho-antibody. Polyclonal AB created by a phospho-peptide and double purified by using both phospho-peptide and non-phospho-peptide will yield a good result.
Calbiochem had published a brochure on phosphospecific antibodies a few years back. They carry a couple of hundred phosphospecific antibodies. You may visit their website www.calbiochem.com