yamshchikov at cber.cber.fda.gov
Sun Sep 10 17:47:02 EST 1995
In article <trotta-180895171009 at mac189.bio.caltech.edu>,
trotta at seqaxp.caltech.edu (Chris Trotta) writes:
> 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
I had the same problems when I needed to concentrate proteins after
deglycosylation. That required dilution of SDS (from the sample buffer) at
ten times before applying the enzyme. Precipitation either with TCA or with
10 vol. acetone resulted in formation of an insoluble pellet which could not be
resuspended back even after prolonged boiling in the sample buffer with 2%
SDS. Partially problem could be resolved by indirect sonication (in a cup)
of the sample prior to boiling. However, I found a better alternative.
Since applying 10 vol. acetone will precipitate almost everything from the
solution (including inorganic salts), addition of sucrose to 5% to the
protein solution prior to precipitation resolved the problem. The pellet,
which is well visible, dissolves easily in the sample buffer even without
heating. The only drawback is salt precipitation if protein was dissolved
in a buffer containing sodium or potassium salts. In my case, for
deglycosylation, I simply use "thinner"
buffer. The procedure, indeed, was adapted from the old sucrose-acetone antigen
preparation method. Hope this helps,
Vladimir F. Yamshchikov.
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