Protein precip. from Guanidine

Kresten 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.
Thank you

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.

Kresten

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">
> <html>
> &nbsp;
> <br>Kresten wrote:
> <blockquote TYPE=CITE>Could anybody please suggest a method for
precipitation
> of protein for
> <br>subsequent SDS-PAGE.
> <p>My sample volume is 200 microliters and contains 40 micrograms of
(pure
> <br>protein). Furthermore the sample is 6.8M in Guanidine
Hydrochloride.
> <p>I have tried to precipitate by diluting with water to 900 ul and
adding
> <br>100 ul 100% TCA w. deoxycholate. I then get a big fluffy pellet,
which
> <br>is much larger than in a sample without guanidine. I tried washing
> the
> <br>pellet with acetone but this completely removed pellet (including
> <br>protein).</blockquote>
>
> <p><br>I've used a similar protocol but I never add
deoxycholate.&nbsp;
> I redissolve the pellet in *ice cold* acetone and respin.&nbsp;
Resuspending
> the final pellet can be difficult if all residual TCA is not
removed.&nbsp;&nbsp;
> I usually add 1/10 volume (of my final resuspension ) in 1M tris pH
7.5
> 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.&nbsp; If you dilute the sample does
> your protein refold?&nbsp; If not you may be able to precipitate just
by
> reducing the denaturant concentration by rapid dilution.
> <pre>--&nbsp;
> Mark Bowen
> Research Associate
> HHMI/Yale University
> Dept. of Molecular Biophysics and Biochemistry</pre>
> &nbsp;</html>
>
>

--
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Kresten Lindorff Larsen, Dept. Yeast Genetics


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