I am expressing a yeast membrane protein 50KDa in E coli. It forms inclusion body so I fused it with MBP.... but now, the protein is too soluble! As it doesn't get pelleted after 100 000 g spin for 1 hour... (conventional isolation of membrane protein is by separating the cytosolic protein from membrane protein by ultracentrifugation).
Does anyone know if membrane proteins can stay in the supernatant after such hard spin without detergent?
Another problem is with solubilistion of another membrane protein. I break E coli open by sonication. Inclusion body and debris is removed by low speed spin. Then membrane protein is pelleted by 100 000 g. I tried to solubilise the membrane protein. The buffer contains 20 mM Tris, 2.5 mM EDTA, 10% glycerol, 1% triton X 100 and various NaCl (30, 100, 250 and 500 mM). After over night incubation, sample containing 100 mM or higher NaCl turn turbid. I tried on different proteins, the same thing happened. Without NaCl, the sample did not turn turbid. I thought it may be the NaCl problem, so I incubated the sample again over night with different NaCl without detergent, all the sample looked transparent. I was wondering it's the combination of Triton X 100 and NaCl caused turbidity?
Does anyone know if Triton X 100 and NaCl are not compatible?
Thanks so much in advance
membrane protein solubilisation
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