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]
