pERK and pAKT in MDA-MB-231 cells by western blot! - (Apr/05/2012 )
I am trying to detect pERK and pAKT in 231 cells by western blot. I am transfecting my cells with siRNA per manufacterer's (siRNA and lipofectamine) instruction, and 24 hours later I replace media with serum free media. Cells are serum starved for 12-14 hours ( = overnight) and then I lyse the cells in lysis buffer containing protease and phosphatase inhibitors and run western blots.
Note: I get good pERK blots and ok pAKT blots. When I ran pERK and pAKT control blots on cells that were in 10% serum media, I got nice bands.
Does this protocol seem correct? Should I be stimulating cells 10-15 minutes before lysing?
Thank you for your help!
It's unclear what you want to study. Do you wish to study the effect of your siRNA knockdown on the basal (serum starved) levels of pERK and pAKT or the serum stimulated phosphorylation of these proteins?