I am culturing SKNSH cells, 1)collecting the cells by trypsin and centrifugation, 2) washing the pellets with pbs.
Then I am adding RIPA buffer to lyse the cells by freeze-thawing. I add protease inhibitors and also DNAse to get rid of DNA precipitate. I am looking for a cell membrane protein.
My buffer recipe is as follows: 40mM TRis (pH 7.6), 120 mM NACL, 1% TritonX-100, .3% SDS & 0.5% Na-deoxycholate.
I add 4x laemlli buffer and boil at 95C for 5 mins. I run gel at 100V and make sure its not hot.
Despite all this my gel is running messy and even beta-actin band shows up broken & mess