TCA Precipitation Problems

[u2528082 at ucsvc.ucs.unimelb.edu.au]

In article <trotta-180895171009 at mac189.bio.caltech.edu>, trotta at seqaxp.caltech.edu (Chris Trotta) writes:
> Hi,
> 
> I have been working with small quantities of a nuclear membrane 
> protein complex.  In order to get enough to analyze, I must TCA
> precipitate.  My problem is after spinning down the protein and 
> acetone washing the pellet, I am unable to resuspend the pellet.
> I have tried resuspending in dH20, buffer containing Triton X-100
> and tris buffer.  No matter what I do to the resuspension, ie heat
> to 65 for a few minutes, add SDS loading dye and heating to 90 for
> 10 minutes, the pellet does not go entirely into solution.  I have
> loaded the pellet in the well and it contains a considerable amount
> of protein, but this results in an unacceptable smear in the lane.
> 
> Does anybody with experience in membrane proteins or protein methods
> have any suggestions for getting around this problem???
> 
> Thank you,
> 
> Chris Trotta
> trotta at seqaxp.caltech.edu


We have been working with membrane proteins from mycoplasma sp. These are very
hyrophobic, and we usually precipitate with ethanol using a carrier of dextran-
mw ca.80,000 @ 10g/ml final conc. The pellet should not be dried- leave wet.
Dissolve in urea-4-6M. This is nonionic and does not interfere with SDS-PAGE or
with ion exchange chromatography.
Let me know if you have probs.
John Walker
U2528082 at hermes.unimelb.edu.au