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Evette, A good machine shop can make a 1 L Amicon-like tirred-cell concentrator quite easily (and many have). But another alternative is the Pellicon Tangential Flow Filtration Cassettes (see http:// www.millipore.com/techpublications/tech1/pb022). The Pellicon XL 50 cassettes (50 sq. cm) work great for most jobs (bringing 1 L down to ~15 mL). This size is not too expensive, particularly if you have an extra peristaltic pump around the lab. The bigger Pellicon 2 Cassettes (0.5 sq. m) are also fantastic for dealing with concentrating proteins secreted into the media; we have taken 16 L down to 400 mL in 45 min. But the startup cost is high for the basic Pellicon 2 system (http://www.millipore.com/catalogue/ module/c558; ~5K about 8 years ago). This larger system can also be used for microfiltration, where we have used it to quickly +16 L insect cells down to ~500 mL for the homogenization step. The downside is that you have to buy an "expensive" cassette for each MWCO and you have to trust your lab mates to keep them clean (as one usually does for FLPC or HPLC columns). The upside is that they perform a lot better than stirred-cell concentrators with less fouling of the membrane. Michael **************************************************************** 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] **************************************************************** 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:[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] >