I have been trying to look at Pyk2 phosphorylation in my cells (human monocyte derived macrophages) for about a year via Western Blot, and am having a massive amount of difficulty doing so. I have been using the CST Phospho-Pyk2 (Tyr402) antibody on whole cell lysates, but the non-specificity of the antibody has made this nearly impossible. I often get different banding patterns and bands that are impossible to analyze, in spite of using the same protocol across all experiments. Recently we have tried to perform IP experiments using the CST total Pyk2 antibody, but we are having difficulties enriching the protein.

I have seen many papers looking at this target and using western blots and IP; however, the methods for many of these are incomplete or do not give enough information on buffers, isolation conditions, etc, for me to try and repeat them. Moreover, papers that perform Western blots on whole-cell lysates do not show the un-cropped blot, so I cannot compare our banding pattern. I have spoken to CST multiple times, but they have not been able to help us address our questions or optimize our workflow. This protein is present and active in our cell line; this has been demonstrated in multiple papers (unfortunately, they are over 10 years old and again, have incomplete methods).

Does anyone have experience working with this target, and a protocol or suggestions to optimize how we do things. Alternatively, are there other ways people have looked at Pyk2 activation (i.e. via IF), and what antibodies/protocols have they used? Any help is greatly appreciated.