Hi, we are currently trying to purify a membrane protein (around 77kDa). We have tried using both denaturing and standard native gels, but the results were ambiguous, in the sense that there is often a protein smear on denaturing gel (probably due to membrane protein aggregation). It also stains weakly in native gels.
We are wondering has anyone tried using Native Page Bis-Tris gel from Life Tech for running membrane protein samples?
It sound like it is your purification step. How did you remove the lipids and solubilize the proteins before running the gel? What was the detergent used? Was this whole cell lysate? If so, how pure was your loading sample and was there a large aggregate of protein still left in your loading well?
We regularly use these NativePAGE gel for blue native analysis of membrane protein complexes. A 77 kDa protein should resolve fine on these gels, too. You just need to be careful when judging the size of your protein based on the native running behavior on these gels because you always look at a protein-detergent-coomassie complex. Just an example: our 160 kDa complex runs at 240 kDa. You may need to try different non-denaturing detergents like DDM, LMNG, DMNG, DM, LDAO, etc to see what works best for your protein.