Need Help in Purification

JT31 jt31 at aol.com
Fri Jul 7 13:29:19 EST 1995


I am currently in the process of purifying an NADP Dehydrogenase from E.
coli. My first procedure is Ammonium Sulfate fractionation....My protein
precipitates at approximately 30% salt conc., however the activity is much
lower (about a 35% loss). Even after desalting through a Sephadex G-10
column there is no increase in activity. Normally after that I put the
semi-crude extract through a Ion exchange process using a DEAE Celluolose
resin. Again the procedure works out find, the enzyme is succesfully
eluted (Using 300mM Tris HCl pH 7.2), However More activity is lost.
Finally After running the extract through a Cibacron Blue resin, and
eluting with approx 1.2M NaCl, the porotein is successfully retrieved but
again more activity is lost! All procedures are performed in a cold room
as well. Does anyone have any hints, tips, or tricks for keeping this
enzyme active??? I would greatly appreciate the Info.

Dominic Ricciardi



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