Affigel chromatography

Stephen Hall shall at bilbo.bio.purdue.edu
Mon Feb 6 18:22:15 EST 1995


In article <3h5f0a$f2t at mserv1.dl.ac.uk>, <kkatrak at nibsc.ac.uk> wrote:

> I'd like some help regarding elution buffers for
> purification of glycosylated proteins on antibody
> coupled affigel-10 columns. The literature says use acid elution or base
elutelution or chelating agents, so which is ideal?
> I only have small amounts of precious material, so
> can't afford to lose any.  Many thanks,


There is no simple answer to the question of an eluting agent.  It is 
empirical and depends on the antigen and your intended use of it.  Does
the antigen need to be antive or can it be denatured?  Are you going to reuse
the column? In general, go from gentle to harsh conditions. In rough order,
easiest to harshest:

      acid
      base
      MgCl2
      LiCl
      Ionic Detergents
      Dissociating agents
      Chaotropic agents
      Organic solvents

In one case, I found acid elution did not work due to hydrophobic
interactions between ligand and protein.  1M propionic acid seems to
work well for me on Affi-Gel 10 columns, but it may not in all cases.

-- 
Stephen Hall
Purdue University Neuroscience Program
Purdue University
West Lafayette, IN 47907-1392



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