G-50 mini columns

Peter pxpst2 at spam.suxs.unixs.cis.pitt.edu
Wed Jun 24 09:27:26 EST 1998


In article <6mr1a8$ctk$1 at nnrp1.dejanews.com>,
theodorn at medlib.georgetown.edu wrote:

> In article
> <Pine.A41.3.96a.980622175559.89572A-100000 at dante09.u.washington.edu>,  "S.
> Tan" <sltan at u.washington.edu> wrote:
> 
> >
> > Hi:
> >
> > I recently purchased ProbeQuant G-50 Micro Columns from Pharmacia Biotech
> > for purification of labeled DNA probes.  I would like to know if it is
> > also possible to use these columns to separate labels from protein
> > preparations.  Specifically, I want to remove free 32P-labeled ATP
> > from a kinase reaction mixture so that I can use the phosphorylated
> > protein product (about 70 kDa) as a substrate for phosphatase assay.  Will
> > I need to re-equilibriate the column with other type buffer?  At what
> > speed should I spin the column at?  Any advice/suggestion would be
> > greatly appreciated.
> >
> > Sincerely,
> > Stan

For Stan:
Seperating proteins by these method depends heavily on the fact that one
molocule will get trapped in the pores while the other molecule is SO
LARGE that it has no chance of entering the pores.  It would be advised
that you beginn by running your column by gravity first before using spin
techniques.  Determine the separation coefficent then go to spin
techniques and monitor columns at different speeds til proper separation
of the molecules occur.  The higher the angular velocity the more likely
you will get the small molecules to exit the pores.  I separate a p98
molecule(scHGF) from free Iodine and I will get free iodine in the protein
peak routinely.

Peter Pediaditakis

-- 
"Don't you eat that yellow snow
            watch out where the Huskies go"    FZ

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