I am over expressing a membrane protein for my current research and i understand that expressing such proteins can sometimes be toxic, therefore i am using strains C43 and C41 to attempt to improve possible yield. However i was just wondering what is the best way to get the protein to effectively solubilise and run on the acrylamide gel to see any expression and then perform western blotting?
Would the use of 8M urea or 0.1% Triton x-100 in the SDS digestion buffer effectively solubilise the protein for SDS-PAGE?
Also i have read boiling at 95 degrees is detrimental as can cause membrane protein aggregation. Any ideas, suggestions or buffer recipes would be greatly appreciated.
Yes, 8 M urea and incubation for 1 h at 37*C instead of 5 min. 100*C are the usual precautions. Note that not all membrane proteins aggregate upon boiling in Laemmli sample buffer. This was originally described for -if memory serves well- an ER ATPase, but I have worked with plenty of bacterial OMP's and have never observed this behavior.
Solubilising them in SDS isn't usually a problem. The Schagger and von Jagow Tricine gel paper (PMID 2449095) incubated at 40C for half an hour before loading rather than the usual quick boil. I've worked on membrane proteins (IMP and OMP) and they've been fine with the standard SDS solubilisation.
Occasionally you may find a membrane protein with multiple lipophilic domains, e.g., with some Archaea, which will induce SDS precipitation due to Sodium incompatibility. If this is the main problem, a solution may be to use Lithium salt of the Dodecyl Sulphate (LiDS) especially if acidity improves the solubility of the polypeptide in question.
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