I am trying to purify a membrane protein using Pichia pastoris expression system. I wish to know what is the best way to prepare whole cell lysates to check for expression
There are simple ways for lysis of pichia to prepare cell lysates for SDS-PAGE. One can also use commercially available lysis buffer (see the link below).
Link: http://www.sigmaaldrich.com/life-science/proteomics/recombinant-protein-expression/cell-lysis/yeast-cell-lysis.html
We usually spin down the cells and resuspend the pellet in sufficient amount of 1x Laemmli buffer, boil it and sonicate (only a few seconds, just to get rid of nucleic acids). Then you can load on a gel.. This is just for checking the expression, making cell lysate for purification is a bit different. Sometimes the sonication in PBS is sufficient, but since you have a membrane protein, it can remain in a pellet after sonication and centrifugation. We resuspend the pellet in buffer with 8M urea and some detergent (2% Triton X-100, 0,1% SDS), then mix together with a sonicate and use for isolation
Hi,
Thank you all for replies. It seems that the membrane protein I am working on is not as problematic as expected. I did the very basic things and it gave me very clear band. I lysed the cells using sonication in a sonication buffer containing PMSF and DTT. I did not boil the sample, but I incubated it with SDS loading buffer and BME at 37 degree Celsius. Thats all but there was gel shifting because of incomplete denaturation I guess. I am going to do a western to confirm it. Otherwise, my gel look excellent with minimal background
Once again, thank you all for your suggestions.
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