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R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [log in to unmask]
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Another question re: Amicon stirred cells...I also seem to recall seeing 1L size stirred cells in older labs of myyouth. My current lab has acquired one of 400 mL, but looking topurchase a bigger one, I can't find any. Any ideas about where we mightfind one?Evette S. Radisky, Ph.D.Assistant Professor and Associate Consultant IIMayo Clinic Cancer CenterGriffin Cancer Research Building, Rm 3104500 San Pablo RoadJacksonville, FL 32224(904) 953-6372 (office)(904) 953-0046 (lab)-----Original Message-----From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf OfGina ClaytonSent: Friday, June 27, 2008 7:37 PMSubject: Re: [ccp4bb] Concentrating proteinHi thereI quite like the Amicon stirred ultra concentration cell systems. Youcan put large volumes in, maximum 1 litre size, I think. As well youcan attach an inert gas such as Argon or Nitrogen, for the gaseouspressure, this reduces oxidation of your sample while itconcentrates. My experience has been that, depending on the filter,the filters are very resistant to various salts even GuHCl, and you getgood recovery. I used to concentrate large volumes of protein down tosay 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 fashionableas they used to be, but perhaps "older labs" tend to have them millingabout somewhere in the back of a cupboard. So most likely you wouldonly have to buy membranes -PM or YM it think depending on you sample.Hope that helpsGinaOn 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 materialwill swell and take up all the liquid except molecules larger thanca.5 kDa.Dear All,we have GCSF protein produced in inclusion bodies. we solubilise itrefold it and then concentrate it using proflux system. still theconcentration of the protein we get is less and volume is more forus to load in Ion exchange chromatography. is there any simpletechnique that can be performed in lab without using any hi-fiinstrument to concentrate the protein in small volume of buffer. theprotein we obtain is about0.7mg/ml and we get 450 ml solution. our column is 110ml lab scale andwe have to work in that only. i have heard of NH4SO4 precipitation.but itrequires 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 anexcellent concentration step as well as a purification methodology.It may take less time and involve less protein loss to pass all thesolution through the IEX column and bind the protein, assuming youhave the protein in a low ionic strength buffer at the appropriate pH.Elution in a smaller volume can be accomplished by increasing the NaClconcentration to an appropriate level. In the bad old days beforebacterial overexpression, we used to to this routinely to concentratea liter or more of protein extract to 50-100 mL after elution from asmall, high-capacity IEX column.Cheers,--------------------------------------------------------------------------Roger S. RowlettProfessorColgate University Presidential ScholarDepartment of ChemistryColgate University13 Oak DriveHamilton, NY 13346tel: (315)-228-7245ofc: (315)-228-7395fax: (315)-228-7935email: [log in to unmask]