HI,

an easy way is to use RIPA buffer (50mM tris ph 7.4, 150 mM NaCl, 1%NP-40, 0.1% SDS, 0.5% Na-deoxycolate, proteases and phosphatases inhibitors). It is extracting almost everything!

good luck

HI,

an easy way is to use RIPA buffer (50mM tris ph 7.4, 150 mM NaCl, 1%NP-40, 0.1% SDS, 0.5% Na-deoxycolate, proteases and phosphatases inhibitors). It is extracting almost everything!

good luck

Hi,

I agree with Matteo, but If you are interested in phosphorylated proteins, I'd recommend to wash your cells twice in PBS, and then lyse them immediately in SDS-PAGE sample loading buffer (1x Laemli). The DTT and SDS will ensure that the phosphorylation status is maximally preserved - if you keep everything on ice, boil as soon as possible and then load them on gel. For some proteins, freeze thaw even in sample loading buffer will result in loss of protein phosphorylation.

Only downside is that the SDS in the loading buffer will interfere with bradford assay, so make sure you lyse about the same amount of cells for all your conditions (and do a blot for a loading control such as actin or tubulin afterwards).

good luck!

Thank you very much!