Affinity chromatography

Dima Klenchin klenchin at facstaff.wisc.edu
Mon Nov 11 18:51:06 EST 1996


In article <23144jda-111196170142 at 35.8.197.48>,
   23144jda at msu.edu (Jenny Davila-Aponte) 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

Many possibilities:

1. Another factor stimulates activity. Try adding flow-through back
to eluted fractions and see if activity is any different. 

2. What is eluent? Affinity (GDP*Mg)? Try higher concentrations and/or
longer elution times - when the affinity for GDP is high, k(off) is 
frequently rate-limiting step. In this case filling the column with 
eluent, stopping the flow for 30-60 min, then eluting helps. 

3. You have an inherently labile protein. Not much can be done here.
Live with it and hope that one day you hit some magic trick that will
stabilize activity. As a matter of fact, I envy you. I've not been able
to push recovery of "my" protein above 5%. 

Good luck, 

- Dima





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