Gel filtration in urea or Gu-HCl?

Helmut Dotzlaw dotzlaw at ccu.umanitoba.ca
Sun Jun 27 13:07:21 EST 1993


Good day, all,

I am currently in the process of attempting to purify a 200 kDa protein
(size determined by SDS PAGE) from human tissue grown in culture.  The
protein is associated with the nucleus and is not soluble unless in the
presence of dissociating agents such as 7M urea or 4M Gu-HCl.

My crude nuclear prep contains few proteins larger than 200 kDa and most of
it much smaller, so I thought a good first approach was to clean it up with
a gel filtration column.  I decided to try a small analytical column, of
Sephacryl S-200 HR, in 7M urea,0.25M NaCl,50mM Tris/Cl pH 7.8.

Column specs:

80x1cm
flow rate=5ml/hr
fraction size=1ml

I ran some standards through the column, 1 mg each of myosin,
B-galactosidase, phosphorylase B, BSA, ovalbumin and carbonic anhydrase, in
a 1 ml sample volume.
When I read OD280, I only got one peak - in the void volume of the column. 
SDS PAGE verified that all proteins eluted in this fraction.  What gives? -
the blurb I have for Sephacryl S200 states that columns can be run in up to
8M urea.  My column had the functional equivalence of a desalting column
;-(.

I did call Pharmacia on Friday to try to talk to the chromatography
specialist, but he was out to lunch - presumably all day or very busy
because he never rang back.

I will try again using 4M Gu-HCl, 50mM Tris/Cl pH 7.5.  A question I have
is can I still use the resin I ran urea through? - I flushed the column
with water and am re-equilibrating with the Gu buffer and will repack it.

Any suggestions/advice most gratefully appreciated - obviously protein
purification is not my forte.

Regards,

Helmut



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