Protein precip. from Guanidine

[Kresten]

In article <7u2l3r$5mt$1 at nnrp1.deja.com>,
  Nick Theodorakis <nicholas_theodorakis at urmc.rochester.edu> wrote:
> In article <7u2caq$ui3$1 at nnrp1.deja.com>,
>   Kresten <kresten at my-deja.com> wrote:
> <snip>
>
> > >
> > > A home-made spin column (G-25 or G-50, e.g.) seems as fast and
> > > easy as
> > > precipitation.
[snip]
>
> Yep. I make them in a 1 cc syringe, with the bottom plugged with a
> porous plastic frit,  glass wool, or a glass bead. Spin for 100-200 X
> g
> for 2 or 3 min., then apply your sample in a volume of. about 75-150
> ul,
> and spin again, the eluate will have the void from the column
> (containing, hopefully, your protein) in the buffer your column is in.
>
> I checked these out pretty thoroughly when I was using a G-50 spin
> column as a binding assay once upon a time (to separate out peptide
> bound to target protein from free peptide). I had less than 1% of free
> peptide come through the column in the absence of protein, and the
> recovery of protein after spin-column was > 90%. Another thing I did
> was
> to check the performance of the column with colored markers. Mix
> phenol
> red with high-MW Blue dextran; after spinning the column, you should
> see
> the phenol red stay in the column and the Blue Dextran elute in the
> void.

Thank you. I might try this out.

>
> One of the things I'm wondering is that maybe your protein becomes
> insoluble after removing the guanidine, leading to poor recovery. If
> that happens, it might precipitate in the column as well. Perhaps you
> could equilibrate the column in urea (8 M?)to keep the protein
> soluble,

Not a problem I think. My protein stock is > 100mg/ml and I think that
unfolding will not lower solubility that much.

Kresten

> but you probably can't use Sephadex - maybe something like P-20 or a
> cross-linked resin like Sephacryl.
>
> 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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