Hi there
I quite like the Amicon stirred ultra concentration cell systems. You
can put large volumes in, maximum 1 litre size, I think. As well you
can attach an inert gas such as Argon or Nitrogen, for the gaseous
pressure, this reduces oxidation of your sample while it
concentrates. My experience has been that, depending on the filter,
the filters are very resistant to various salts even GuHCl, and you
get good recovery. I used to concentrate large volumes of protein
down to say 50-25ml then switch to the same system, in a much smaller
cell i.e. 10ml, to get down to 1-2ml. And they are fairly fast too.
I get the impression, perhaps incorrectly, they are not as
fashionable as they used to be, but perhaps "older labs" tend to
have them milling about somewhere in the back of a cupboard. So most
likely you would only have to buy membranes -PM or YM it think
depending on you sample.
Hope that helps
Gina
On Jun 27, 2008, at 9:19 AM, Roger Rowlett wrote:
Guenter Fritz wrote:
A mild and quick method is to use dry Sephadex G-25. The material
will
swell and take up all the liquid except molecules larger than ca.
5 kDa.
Dear All,
we have GCSF protein produced in inclusion bodies. we solubilise
it refold
it and then concentrate it using proflux system. still the
concentration
of the protein we get is less and volume is more for us to load
in Ion
exchange chromatography. is there any simple technique that can be
performed in lab without using any hi-fi instrument to
concentrate the
protein in small volume of buffer. the protein we obtain is about
0.7
mg/ml and we get 450 ml solution. our column is 110ml lab scale
and we
have to work in that only. i have heard of NH4SO4 precipitation.
but it
requires protein conc more than 1 mg/ml.
kindly help me to progress in my experiment.
One of the beauties of ion-exchange chromatography is that it is an
excellent concentration step as well as a purification methodology.
It may take less time and involve less protein loss to pass all the
solution through the IEX column and bind the protein, assuming you
have the protein in a low ionic strength buffer at the appropriate
pH. Elution in a smaller volume can be accomplished by increasing
the NaCl concentration to an appropriate level. In the bad old days
before bacterial overexpression, we used to to this routinely to
concentrate a liter or more of protein extract to 50-100 mL after
elution from a small, high-capacity IEX column.
Cheers,
--
----------------------------------------------------------------------
--
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]
