Hi all,
I am doing western blot for acetylated p53(K382) in SHSY5Y cells.
I have tried several changes but i am not getting bands in SHSY5Y cells and every time I am getting band in positive control.
Has any one done western blot for acetylated p53 in SHSY5Y cell ? Kindly help me.
Treat cells with 1uM TSA and 5mM nicotinamide along with dox will help. Make sure your lysis buffer contain 10 uM TSA and 5mM nicotinamide to prevent lost of p53 acetylation during lysis. Check my 2001 cell paper for detail. Good luck!
Agree with Luo's comment. By the way, do you mean Hela cells? Since HPV protein in Hela cells can degrade p53, endogenous p53 in Hela cells is usually not a good positive control. I am suspicious of your antibody. In my hands, a Genetex K382Ac antibody is very good.
thank you Jianyuan Luo and Weijia Cai for you valuable suggestions.
My concern is p53 acetylation in SH-SY5Y cells. anyways I am using the antibody from cell signalling and they have used doxo treated hela cells so, I have used the same as positive control.
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