G-50 mini columns

S. Tan sltan at u.washington.edu
Mon Jun 29 13:48:44 EST 1998



Thank you for all for responding to my inquiry and your helpful
suggestions and advice.  I shall try G-50 columns first since I have them
in the lab, and let you know how it works out.  Thanks again --Stan.

On Sat, 27 Jun 1998, Dima Klenchin wrote:

> In article <pxpst2-2406981427260001 at pelli.pathology.pitt.edu>, pxpst2 at spam.suxs.unixs.cis.pitt.edu (Peter) wrote:
> 
> >> > 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.
> 
> >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.
> 
> It's a matter of quantity, isn't it? I separate proteins from iodine and 
> FITC by spin gel-filtration on Bio-Gel 6 and get no more than 2% 
> contamination. When you have 30 samples (for buffer exchange, for example), 
> that's the only way to go. So yes, playing with centrigugation speed/times
> in advance does help, but the technique is quite valid. G-50 is not 
> universally useful for proteins, but G-25 and Bio-Gel 6 (the latter has 
> slightly lower non-specific binding in my experience) are fool-proof.
> 
>         - Dima




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