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
-
