GDP Affinity Chromatography

[Dr E. Buxbaum]

deroche1 at pilot.msu.edu (Amy DeRocher) wrote:
>I am trying to purify a fucosyltransferase that is involved in cell wall
>synthesis in higher plants, and one of the steps of my purification
>strategy is a GDP affinity column step.  This step gives me good
>enrichment, but the recovery of activity is mediocre (15 - 20%).  Obtaining
>tissue for protein extraction is laborious, so I would like to improve my
>recovery of activity.  I have tried changing several ingredients of my
>column buffer: buffer, pH, detergent, divalent cations, ionic strength, and
>stabilizing agents; but have not been able to improve my recovery.  The
>column buffer that I am currently using contains 20% glycerol, 50 mM pipes
>KOH pH 6.2, 100 mM KCl, 0.1% decylmaltoside, aprotinin, caproic acid,
>leupeptin, and pepstatin.  I would appreciate any advice or useful
>suggestions.

First you have to find out where the loss occurs. Is binding to the 
column incomplete (protein appears in the flow through)? Does the protein 
stick to the column (amount of your protein (not activity!) coming out of 
the column is less than what you put on, determined by ELISA or similar)? 
Or does your protein lose activity during purification (recovery of 
protein is complete, but activity is to low)?

Once that question is answered, the solution should be simple.