Protein precip. from Guanidine

Kresten kresten at my-deja.com
Wed Oct 13 08:28:01 EST 1999


In article <7u0vcq$ttn$1 at nnrp1.deja.com>,
  Nick Theodorakis <nicholas_theodorakis at urmc.rochester.edu> wrote:
> In article <3803d94b.356044020 at news>,
>   jrt at home.com (John Thompson) wrote:
> > Kresten <kresten at my-deja.com> wrote:
> >
> > >Could anybody please suggest a method for precipitation of protein
> > >for
> > >subsequent SDS-PAGE.
> > >
> > >My sample volume is 200 microliters and contains 40 micrograms of
> > >(pure
> > >protein). Furthermore the sample is 6.8M in Guanidine
> > >Hydrochloride.
> > >
> >
> > Why precipitate at all. You can probably add 5X sample buffer and
> > load
> > the sample directly on SDS Page.  If you need to concentrate to I'd
> > suggest a low mw cutoff spin filter (try Amicon or Millipore) that
> > will allow you to change the buffer and reduce the volume at the
> > same
> > time.
> >
> > Yours,
> > John Thompson
> > Merck Research Labs
> >
>
> The problem will be the ionic content of the guanidine, which will
> surely mess up the electrophoresis. It wouldn't be a problem with urea
> (but I would skip the boiling step). Is there a way the the original
> poster could dialyze against urea to remove the guanidine (assuming a
> denaturant was needed to keep it in solution)?

The idea behind the experiment was simply to modify the protein
chemically and check for modification by SDS-PAGE. So in principle I
could dialyse against water/buffer (after modification) but I would
prefer something more simple like TCA prec. since I have several
samples.

The protein denatures around 5M Guanidine so I do not think it can be
substituted by urea although I haven't specifically tested this.

I've also tested denaturing in 5% SDS and subsequent TCA prec. in a 4x
diluted (that is 1.25% SDS) sample. No pellet. Hmm.

Perhaps I should try to load directly on the gel.

Thank you
Kresten

>
> Nick
>
> --
>  _______________________________________________
> | Nick Theodorakis                              |
> | nicholas_theodorakis at urmc.rochester.edu       |
> | (previously theodorn at gusun.georgetown.edu)    |
>
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>

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Kresten Lindorff Larsen, Dept. Yeast Genetics


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