Cell Signaling - (Mar/17/2006 )
I am looking for other methods to show that I have complexes binding to HUVEC Cells. I am wanting to submit a paper but feel that confocal microscopy, ELISA and western blots will not be enough and not to mention my PI does not lik Flow so are there any other methods to show phospholyated protein activation such a pERK, JNK, p38, GSK etc.
I dont think I understood. Are you looking for methods to show phosphorylation of a protein? This can be done by an immunoblot (aka Western) or by a kinase assay. And If you are looking for methods to show direct binding of 2 proteins, well, co-IP sounds good to me, as well as crosslinkers maybe.
Even me wanna know.. by western u mean.. with antibodies against the phosphorylated form of the protein? and could u plz elaborate on the crosslinkers bit?
u get phospho-specific antibodies to the protein of ur interest. You have to first probe with the P-antibody followed by the T-antibody. For eg. If you are looking to see phosphorulated form of JNK, first probe the blot with p-JNK and then with T-JNK.