blue dye column

Federici federici at icgm.cochin.inserm.fr
Fri Jan 24 04:30:19 EST 1997


In article (Dans l'article)
<01bc02b5$0d4fc900$0815f88f at hskim.kaist.ac.kr>, "Jang Young Lee"
<jylee at bioneer.kaist.ac.kr> wrote (écrivait) :

> 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

the column may be saturated; it depends on how you carry out the
purification. Is the blue dye column the first step of the purification
procedure ? In this case, other enzymes may have a higher affinity for the
blue cibacron and so compete with the dehydrogenase that you want to
isolate. You can add a pre purification step: for instance, gel filtration
or ion exchange chromatography in order to obtain a fraction able to be
eluted specifically from the blue dye column.



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