Non-lysis of stromal and bone marrow cells for IP
I need to perform IP from the membrane proteins expressed on S17 and bone marrow stromal lines. I use the buffer for lysis with HEPES, NaCl, MgCl2 and 1% lubrol. I incubate this along with the cells, in the presence of protease inhibitors and PMSF, for one hour at 4 deg for lysis. But I am not able to completely dissolve all the membrane bound proteins into the lysate. The lysis is not efficient or completely failed. I have also been asked not to change the concentration of lubrol or even the detergent itself. For ex. I should not use harsher detergents like triton x-100 or SDS to lyse the cells. My colleagues get good lysis but I cannot even get it done. Please help me in solving this problem. I am in very much need of a solution.
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