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:[log in to unmask]] On Behalf Of

Gina Clayton

Sent: Friday, June 27, 2008 7:37 PM

To: [log in to unmask]

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: [log in to unmask]