Protein precip. from Guanidine

Kresten kresten at my-deja.com
Wed Oct 13 11:34:35 EST 1999


In article <7u29qn$sk0$1 at nnrp1.deja.com>,
  Nick Theodorakis <nicholas_theodorakis at urmc.rochester.edu> wrote:
> In article <7u21ch$li6$1 at nnrp1.deja.com>,
>   Kresten <kresten at my-deja.com> wrote:
> <snip>
>
> >
> > The protein denatures around 5M Guanidine so I do not think it can
> > be
> > substituted by urea although I haven't specifically tested this.
> >
>
> Do you mean under or over 5 M Guaninidine?

The transition from native to denatured starts around 4M and is more or
less finished around 6M. In a two-state model I get following values:
[GuaHCl]50% = 4.9 +/- 0.1 M
m-value = 9 +/- 1 kJ/(mol*M)

>
> > 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.
>
> Let us know if that works. With that much salt, I betcha it won't even
> run into the gel.

I mean with the SDS denaturation.

>
> A home-made spin column (G-25 or G-50, e.g.) seems as fast and easy as
> precipitation.

I've thought of that but I do not know how to check where the protein
is unless I either use more protein and check by A280 or less protein
but run an SDS-gel.

Could I simply make the coloumn (G-25), equilibrate w. water, add
sample, spin everything through and assume that most salt will stay on
the column?

Kresten

>
> Nick
>
> --
>  _______________________________________________
> | Nick Theodorakis                              |
> | nicholas_theodorakis at urmc.rochester.edu       |
> | (previously theodorn at gusun.georgetown.edu)    |
>
> Sent via Deja.com http://www.deja.com/
> Before you buy.
>

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Kresten Lindorff Larsen, Dept. Yeast Genetics


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