Cibacron blue ourification of ADH?

MG mavigozler at yahooDOT.com
Fri Sep 27 13:53:47 EST 2002


20 mM Tris?  what pH?  Counter-ion?
Also make sure your EDTA is what it is...10-fold lower than magn. chloride.
You don't mention whether you are measuring activity (or looking at your
protein
in gels) in both the effluent (unbound) as well as eluate (bound).  When the
latter
doesn't contain what you expect, always check the former, as well as your
loading material.

"Fergus Doherty" <Fergus.Doherty at nottingham.ac.uk> wrote:
> Has anyone experience of purification of yeast alcohol dehydrogenase using
> the dye ligand Cibacron Blue F3GA?  I have tried it many times without
> success.  Start/binding buffer 20mM Tris, 5mM mercaptoethanol, 5mM MgCl2,
> 0.4mM EDTA, as described in several protocols but ADH doesn't bind!
Plenty
> of ADH activity.  Any help appreciated as lab class approaches!





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