Hi there
I actually just consulted, about your question, with one of the
longer term members of the department about this. And we came to the
conclusion that 4- 500ml was probably the maximum size.
Gina
On Jun 30, 2008, at 11:15 AM, Radisky, Evette S., Ph.D. wrote:
Another question re: Amicon stirred cells...
I also seem to recall seeing 1L size stirred cells in older labs of my
youth. My current lab has acquired one of 400 mL, but looking to
purchase a bigger one, I can't find any. Any ideas about where we
might
find one?
Evette S. Radisky, Ph.D.
Assistant Professor and Associate Consultant II
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372 (office)
(904) 953-0046 (lab)
-----Original Message-----
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Gina Clayton
Sent: Friday, June 27, 2008 7:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Concentrating protein
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]
