Need Help in Purification

Joe Mack mack at ncifcrf.gov
Fri Jul 7 14:15:52 EST 1995


In article <3tjudv$qp8 at newsbf02.news.aol.com> jt31 at aol.com (JT31) writes:
>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). 

This is normal in my experience.

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

Wouldn't worry about it - recovering 10% of activity after 3 steps happens
a lot. As you get more experience with the protein, you might get better
yields, but for now as long as the enzyme is being purified, then there
may not be much you can do.

	Joe Mack mack at ncifcrf.gov




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