Protein separations using Ion Chromatography

Giovanni Maga maga at vetbio.unizh.ch
Tue May 28 07:55:49 EST 1996


In article <31A5F63C.1C12 at rutchem.rutgers.edu>, hall at RUTCHEM.RUTGERS.EDU
("G.S. Hall") wrote:

> We want to separate proteins, albumin, cerulloplasmin, transferrin, and 
> myoglobulin from human serum.  Tried GPC but resolution can not separate 
> transferring and albumin.  I heard that ion chromatography using 
> gradients works well.  What type of column would you recommend?
> 
> Thanks in advance,
> 

In general, since both albumin and transferrin are quite well
characterized proteins, you can look up the pI of each. Then, having
established which pH will allow you to obtain the maximal difference in
protein charge, you can choose the appropriate cation- (binds positively
charged proteins) or anion- (binds negatively charged proteins) exchanger.
For example, if the two proteins of interest will have pIs of,
respectively, 6.0 and 7.0, at pH 6.5 one will be negatively charged and
the other positively charged. Thus, peforming a chromatography with
Q-sepharose at pH 6.5 should elute the positively charged in the FT and
the negatively charged at low salt concentration. Alternatively, at pH
7.1, both will be negatively charged, but the first much more than the
second (1 pH point above the pI vs. 0.1). Thus a salt gradient on a
Q-Sepharose should separate them. This is how it goes. If you do not know
the pI and do not want to determine it by IEF or Chromatofocusing (which
is a bit boring), you can just run a cation- and an anion-exchanger column
in parallel at pH 7.5 with a NaCl gradient from 10 mM to 0.7 M (just a
typical starting fractionation) and look what happens. By determining the
elution points of both proteins on both columns, you should be able to
design the conditions of chromatography for a real separation. Good
starting columns for ion-exchange chromatography are Q-Sepharose (anion
exchanger) and S-Sepharose (cation exchanger) from Pharmacia. You can buy
the matrix and then fill up any kind of column of the volume you like
(even plastic syringes are good at low flow rates).
Hope it helps. 
G.Maga.



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