yes, but you have to check fisrt the protein doesnt crash out in 1 M (NH4)2SO4 or whatver concentration needed.. problem with amm. sulfate is that its good at salting-out proteins also, which we of course know... (which is of course another way to concetrate your protein, but there are more gently ways.), -----just to add the cautinary note there.
i would go with e.g. the large vol centripreps (amicon/vivaspin whatever) /IEX and dialysis or desalting columns if needed. in whatever combination needed. Best, Tommi Quoting Roger Rowlett <[EMAIL PROTECTED]>: > There are actually two very good ways to concentrate dilute protein > while carrying out a purification step. If the solution is low ionic > strength, then IEX is the way to go. If the solution is too high in > ionic strength to do IEX directly, then add ammonium sulfate (usually to > > about 1.0 M) and bind protein to a hydrophobic interaction > chromatography medium. We usually use butylsepharose for HIC > purification. It will be necessary to work out conditions for both > binding and elution before committing the entire sample, of course. > > 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] > > Tommi Kajander wrote: > > 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: [email protected] > >>> > >>> 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: [email protected] > >>> 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 > > > > - > > > -- 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
