Membrane Protein SDS PAGE and WB - (Jan/11/2013 )
I am trying to do western blotting of highly hydrophobic membrane (has 11 TM stretches) protein fused with MYC and GFP tags transfected in various mammalian cell lines. I have tried all options of western blotting used for hydrophobic membrane proteins with no results.
I am only able to see the smear and blob at the top of well/WB membrane and no band at expected size with boiling from one minute to 10 minutes.
As membrane proteins tends to get aggregate upon boiling. I tried to do various incubations in 2xSDS sample buffer as well as 2x Urea SDS sample buffer starting at room temperature to 37, 60, 70, 80 and 90 C. But I am not able to see band of my protein. While I am able to see the smear or blob at top even with one minute of boiling. So as suggested by experts and literature I tried incubations at all other lower temperatures, but I am not able to detect the band.
I have tried RIPA and 8M Urea CHAPS lysis buffers followed by standard 2X SDS samples buffers as well as 2xSDS buffer with 8 M urea.
In the normal scenario, membrane proteins WB usually work without boiling but I have no success.
Pl. let me know if you have any suggestion for this problem.
Did you do Coomassie Staining or Ponceau Staining whether its there or not? check this, Which membrane aere using for the transfer? Its maybe just a technically problem.