G-50 mini columns

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Sat Jun 27 16:32:53 EST 1998


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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