Protein precip. from Guanidine
kresten at my-deja.com
Wed Oct 13 11:41:40 EST 1999
First, please do not post in html since this hardens reading the msg.s.
I tried diluting Guanidine w.o. succes.
Maybe I should try to leave out the deoxycholate. As far as I understand
it should help bring down the protein - acting as a "carrier". But it
could of course also bring down the guanidine.
I tried to wash the guanidine-holding TCA-precipitate w. acetone.
Everything was dissolved including protein.
In article <38049C6F.CDDD95FA at laplace.csb.yale.edu>,
Mark Edward Bowen <mb at laplace.csb.yale.edu> wrote:
> <!doctype html public "-//w3c//dtd html 4.0 transitional//en">
> <br>Kresten wrote:
> <blockquote TYPE=CITE>Could anybody please suggest a method for
> of protein for
> <br>subsequent SDS-PAGE.
> <p>My sample volume is 200 microliters and contains 40 micrograms of
> <br>protein). Furthermore the sample is 6.8M in Guanidine
> <p>I have tried to precipitate by diluting with water to 900 ul and
> <br>100 ul 100% TCA w. deoxycholate. I then get a big fluffy pellet,
> <br>is much larger than in a sample without guanidine. I tried washing
> <br>pellet with acetone but this completely removed pellet (including
> <p><br>I've used a similar protocol but I never add
> I redissolve the pellet in *ice cold* acetone and respin.
> the final pellet can be difficult if all residual TCA is not
> I usually add 1/10 volume (of my final resuspension ) in 1M tris pH
> and vortex before I add 1 x SDS buffer.
> <p>I've wondered if diluting the sample before TCA precipitation would
> help reduce the Gnd precipitation. If you dilute the sample does
> your protein refold? If not you may be able to precipitate just
> reducing the denaturant concentration by rapid dilution.
> Mark Bowen
> Research Associate
> HHMI/Yale University
> Dept. of Molecular Biophysics and Biochemistry</pre>
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Kresten Lindorff Larsen, Dept. Yeast Genetics
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