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Membrane Protein SDS PAGE and WB

Membrane Protein SDS PAG

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#1 yalegenetics

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Posted 11 January 2013 - 12:07 PM

Hi Experts

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.

#2 Pangea

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Posted 12 January 2013 - 07:39 AM

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.




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