1. collect cells and wash with PBS once.
2. add RIPA buffer and 4C rotate for 30 min
3. 15000rpm, 4C for 20 min
4. store the supernatant at -80C.
50mM Tris-Cl, pH8
1% Triton X-100
I add protease inhibitor cocktails in PBS and RIPA before use.
As you may know, T cells have very big nuclei and quite limited cytoplasm. The problem is after rotation at 4C for 30 min, for some T cell lines, all nuclei were disrupted. I have checked that by microscopy. But for the others, I can see big debris (either viscous or not) by nake eye and many remaining nuclei by microscopy. I tried sonication and that did get rid of the debris for me. However, the IB result didnot look good with the sonicated samples. So I really want a protocol to completely lyze the cells without sonication or other extra procedures. My target protein localizes in both cytosol and nucleus and I want to compare the expression level between these cell lines.
Thank you very much.
Edited by gyma, 25 July 2011 - 05:42 AM.