phosphate + freeze thaw vs RIPA buffer for cell lysis - (Sep/20/2011 )
I am just wondering if anyone has ever extracted protein with simple phosphate buffer pH 7.4 with 1 mM EDTA, and I freeze thaw 3 x to lyse them and run them for western blotting? Is that acceptable at all? I have read abcam's guidelines for lysing cells and it appears that RIPA or NP40 buffers are being used with 30 minutes agitation in chilled condition. I don't know how different it would be if I start lysing my cells with RIPA buffer or NP40 buffer instead..
It will work for the particularly soluble proteins. The detergent steps help solubilise (some of) the poorly soluble proteins and degrade fats etc. that are found in the membranes.
Freeze/thaw cycles can also result in the degradation of the proteins in the samples.
Thanks Bob1. I am trying RIPA buffer now to lyse my cells.