Jang Young Lee wrote:
>> Currently, I am working with a cibacron blue dye columne to purify a
> dehydrogenase of my interest. The enzyme does not show activity without
> NAD. As far as I know, cibacron blue has a relatively strong affinity
> towards NAD requiring enzyme. To the contrary, the activity was found in
> non-bound fractoin in my case. Can anyone give me a reasonable explanation
> on this.
> I'd also like to know the optimized elution strategy for this dye column
> (although I know that it depends on the protein.).
>> Thanks.
>> Jang Lee,
>jylee at boineer.kaist.ac.kr
Although Cibacron Blue (and many other dyes) does bind NAD(P) enzymes rather better than most proteins, there
are many exceptions. It helps if the enzyme also has a high isoelectric point, so that cation exchange
effects add to the interaction. A low I.E.P. NAD-dehydrogenase is certainly not guaranteed to bind to
Cibacron Blue, but it can also depend on the buffers used. Try a lowish pH (say 6.0), with some transition
metal ions present e.g. 2 mM Mn2+. Elution preferably stepwise by increasing ionic strength and/or pH.
