couldnt agree more.. just pump the dilute solution through the ion exhange column.. or was there salt in it to prevent binding?
or what was wrong with just using 80 ml centripreps or equivalent? not that high-tech, all you need is a regular 250 ml centrifugre tube rotor... (well the centrifuge also). tommi Quoting Phoebe Rice <[EMAIL PROTECTED]>: > A layer of dry PEG on the outside of the dialysis tubing > works too, without changing the ionic strength. But you'll > probably get small molecular weight contaminants from the > PEG entering the bag, so you'll have to dialyze against real > buffer afterwards. The whole procedure in the end might be > more annoying than just loading a very large volume onto an > ion exchange column. > Phoebe > > > ---- Original message ---- > >Date: Fri, 27 Jun 2008 10:16:53 -0500 > >From: "First, Eric" <[EMAIL PROTECTED]> > >Subject: Re: [ccp4bb] Concentrating protein > >To: CCP4BB@JISCMAIL.AC.UK > > > > Back in the old days, we used to put the protein in > > dialysis tubing and surround it with solid NaCl (a 2 > > liter graduated cylinder works well for this). > > After several hours, rinse off the outside of the > > dialysis tubing, readjust the dialysis clips and > > remove excess tubing, and dialyze against your > > favorite buffer. I suggest wrapping parafilm around > > the ends of the dialysis clips, as the dialysis > > tubing will swell when you dialyze out the NaCl. > > You lose some protein due to adhesion to the > > dialysis tubing, but it works fairly well. > > > > Eric First > > Dep't of Biochemsitry and Molecular Biology > > LSU Health Sciences Center in Shreveport > > > > -----Original Message----- > > From: CCP4 bulletin board on behalf of Exec > > Sent: Thu 6/26/2008 10:28 PM > > To: CCP4BB@JISCMAIL.AC.UK > > Subject: [ccp4bb] Concentrating protein > > > > 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. > Phoebe A. Rice > Assoc. Prof., Dept. of Biochemistry & Molecular Biology > The University of Chicago > phone 773 834 1723 > http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 > > RNA is really nifty > DNA is over fifty > We have put them > both in one book > Please do take a > really good look > http://www.rsc.org/shop/books/2008/9780854042722.asp > > -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
