Elution Conditions from Affinity Resin??
Marcus, Dr J.
J.Marcus at botany.uq.edu.au
Wed Sep 27 17:53:14 EST 1995
Stephen Gately wrote:
> I would be interested in any protocol for eluting from an affinity column
> other than increasing NaCl concentrations. Unfortunately the enzyme I am
> eluting is inactivated by increasing salt concentrations.
> Any response will be appreciated.
I have not done that much affinity chromatography but
I think there are several things you can try to get your
protein to elute from an affinity column. I am not sure
what type of affinity column you are using but here are
1) If you are dealing with an enzyme, try adding substrate
or product. An example of this would be the elution of a
dehydrogenase by addition of NAD+.
2) Raise the pH of your elution buffer; perhaps even a pH
gradient will work for you. Of course you'll
have to see what effects that has on your protein.
3) If you are using a column with some type of immobilized
ligand, you could always try adding some of the same ligand
to the elution buffer (i.e. non-immobilized ligand). THis
point is similar to #1 but differs in that I am refering to
the dye iltself rather than a natural ligand.
4) Lastly, you might consider trying another affinity
column with lower affinity toward your protein. This will
allow elution under conditions that are not so harsh.
All the best,
John Marcus Marcus at tpp.uq.edu.au (Dr J.Marcus)
Cooperative Research Centre for Tropical Plant Pathology
5th Level John Hines Building
University of Queensland
St. Lucia, QLD 4072
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