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Hello everyone, I am working on a yeast membrane protein, about 100 KDa. The protein is expressed as an inclusion body in E coli even at 18 C. The problem is solved when MBP is fused to the N terminus of the protein. The resulting protein is ~140 KDa, pI 6.0. The expression level is moderate. After cell disruption and fractionation by high speed spin (100K g), the protein land in the pellet fraction as expected for a membrane protein. The pellet is then solublised in various conditions and assessed for solubility. Buffer I used: 50 mM phosphate pH 7.5, 250 mM NaCl, 5% Glycerol With different detergents at final concentration of - 0.1% ~ 4% Trition X 100 - 4% OG - 2% DDM - 2% DM - 4% CHAPS - 2% C8En - 2% C12En - 2% Cymal-4,-5,-6 - 2% MEGA-10 - 2% HEGA-10 - 2% SDS Only SDS can completely solubilise the protein, other detergents solublise less than 10%... Can anyone offer some advice? really appreciated Kien