Elution Conditions from Affinity Resin??

[Marcus, Dr J.]

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.
> 

Hi Stephen,
     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 
several suggestions:

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




_________________________________________________________
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
AUSTRALIA
Fax: 61-7-365-4771
Phone: 61-7-365-4764