I have been trying for weeks to western blot a 360kDa membrane protein. I've followed suggestions from online posts but I haven't been successful.
I have prepared 3 different lysates (Thermo Scientific M-per and Mem-Per kits and one using RIPA buffer) using three different cell lines (Two of which should have this protein of interest).
I am currently using a 4-15% gradient gel, I load about 60 ug of my samples, run the gel at 150V for 2.5 hrs, transfer onto nitrocellulose membrane over night in tris-glycine based buffer with 0.05% SDS and 20% methanol overnight at 30V and then for 1 more hr at 70V. (I've also tried other transfer times and voltages as well as transfer buffer with only 0.037% SDS)
Staining of my membrane after transfer with Ponceau stain does not show bands at the expected location but I have also stained my gel once after transfer and no bands are present at the expected location either.
Can anyone please offer any suggestions?? I am at a dead end.