Hi, I'm having trouble in western blot for kinase (Ca2+/calmodulin-dependent protein kinase II ) and a phoshatase (calcineurin a) proteins. Those proteins don't appear in membrane. I already tested different protein concentration, extraction protocols (protease inhibitor cocktail SIGMA, direct lysis in laemmli buffer), different transfer conditions (overnight, 1h, 2h...). So, I would to know if exist something unique for detect that kind of proteins.
Thanks.
To eliminate a number of possibilities that might be contributing to the outcome, one would need more information- (i) do you have positive and negative controls; (ii) are antibodies from a reliable source and reported to work in immunoblotting for your proteins isolated from cells from a species you are interested in; and (iii) do you have problems with only these proteins (and other proteins are detectable using your protocol)?
To eliminate a number of possibilities that might be contributing to the outcome, one would need more information- (i) do you have positive and negative controls; (ii) are antibodies from a reliable source and reported to work in immunoblotting for your proteins isolated from cells from a species you are interested in; and (iii) do you have problems with only these proteins (and other proteins are detectable using your protocol)?
Need to have a positive control to varify your antibody is working.
I take it you are detecting total proteins and not phosphorylated? I would guess it has something to do with improper lysis. Triton or NP based lysis buffers have worked for me with manual tissue grinding. Is your loaded total protein amount sufficient?
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