I have samples of KO membrane proteins in Hela cells that I am trying to view on WB and am having trouble determining the best lysis buffer to do so. We have tried 0.5% Np-40, 10mM Hepes pH 7.4, 100 mM NaCl. We have also tried this same buffer with added 0.1% SDS which seemed to give better protein concentrations. It was recommended to use 1% SDS for whole cell lysate which we tried in place of the NP-40 and found that the SDS tended to precipitate in any ice. Any suggestions? Thanks!
If you're using lysate samples only for western blot or you don't need any functional activity, then you can be more aggressive with your lysis conditions. NP40 is a nonionic detergent and could be too mild for isolating membrane protein targets, so RIPA (containing ionic detergents) will probably be more suitable.
You should also consider performing the lysis at room temperature. Make sure you include the appropriate protease inhibitor cocktail and a 30' in RIPA buffer will perform well. I don't have an exact number, but SDS precipitates pretty easily at lower temperatures.
Good luck, keep testing!
Do you want a whole-cell lysate for your WB, or do you want to isolate membranes and then WB only those?
I have described the isolation of plasma membranes in https://www.researchgate.net/post/How_to_extract_cell_membrane_proteins_from_cell_culture_specifically_ABC_family_Can_somebody_suggest_how_can_I_get_good_yeild.
If you want a whole-cell lysate, RIPA-buffer is commonly used, and I would at least start with that.
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