I take the cells trypsinize the cells and wash them with cold PBS two times.
Then i use RIPA lysis buffer according to the pellet and add protease inhibitors 1/20th volume of lysis buffer. Pipet it for mixing and then incubate on ice for 15 mins. then i sonicate it for 3 cycles of 20 seconds each.
Further i centrifuge at 14000g for 30 mins at 4C.
and then take the supernatant and make aliquots.
and i am getting these type of gels.
Thanks
Yep. Do not leave empty wells. You are most likely not having enough sds in your sample buffer.
I would avoid trypsinizing the cells. I would scrape them or use EDTA to release them from the surface, after first washing them. Trypsin will cause proteolysis of external parts of proteins. Moreover, since you add RIPA buffer to lyse the cells before you add protease inhibitors, residual trypsin remaining after the washes could cause proteolysis of intracellular proteins.
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