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 <[log in to unmask]>:
> 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: [log in to unmask]
>
> 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 <[log in to unmask]>:
> >
> >
> >> 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" <[log in to unmask]>
> >>> Subject: Re: [ccp4bb] Concentrating protein
> >>> To: [log in to unmask]
> >>>
> >>> 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: [log in to unmask]
> >>> 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
|
