Dear Colleagues,
we would like to evidence differences in acetylation of p53 in HUVEC cells by western blot or flow cytometry, could you provide us with a good protocol?
Thank you very much
Hi Sarah,
how about a pull down of a modified RIPA lysate (remove SDS from classical RIPA) with a pan anti-acetyl (from Cell Signaling) and then make a p53 blot on the eluate..
Best regards
Andrei
IP-western approach is not likely to work as noted by Andrei. This is because IgG heavy chain co-migrates with p53 protein band, thus making it difficult to immunodet p53. Instead, antibody to acetylated lysine, which is covalently linked with agarose, should be used and the bound proteins need to be eluted using 0.5 N HCl to dissociate antigen (acetylated-p53) from antibody. The eluate can be analyzed by immunoblotting with appropriate negative and positive controls.
You could do a pull-down, but as Divaker said, you run into the problem that p53 co-migrates with IgG heavy chain, so IgG will cover up the p53 signal. I haven't done this myself, but I know people in my lab have run into this problem, and there are special kits you can use to get around it. I think our lab uses the TrueBlot system from Rockland, which only detects non-denatured immunoglobulins, so it doesn't detect the denatured IgG from the pull-down.
http://www.rockland-inc.com/TrueBlot-IP-Western-Blotting-Main.aspx
Thank you very much for sharing...Any specific inhibitors to add in the lysis buffer?
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