Protein precip. from Guanidine
nicholas_theodorakis at urmc.rochester.edu
Wed Oct 13 14:04:38 EST 1999
In article <7u2caq$ui3$1 at nnrp1.deja.com>,
Kresten <kresten at my-deja.com> wrote:
> > 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?
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
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,
but you probably can't use Sephadex - maybe something like P-20 or a
cross-linked resin like Sephacryl.
| Nick Theodorakis |
| nicholas_theodorakis at urmc.rochester.edu |
| (previously theodorn at gusun.georgetown.edu) |
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