Affinity chromatography

[Amy DeRocher]

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



-- 
Amy DeRocher
MSU-DOE Plant Research Laboratory
Michigan State University
deroche1 at pilot.msu.edu