blue dye column

Warren Gallin wgallin at gpu.srv.ualberta.ca
Wed Jan 15 12:51:22 EST 1997


In Article <5bj2a0$3l5u at news.doit.wisc.edu>, klenchin at facstaff.wisc.edu
(Dima Klenchin) wrote:
>In article <01bc02b5$0d4fc900$0815f88f at hskim.kaist.ac.kr>, "Jang Young Lee"
<jylee at bioneer.kaist.ac.kr> 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.
>
>The reasonable explanation is that dye chromatography remains totally 
>empirical and all the general outlines you can find in books (Blue -> NAD, 
>Red -> ATP, etc) reflect only a _tendency_ for proteins that do bind. 
>Play with Mg2+ concentration ot try other dyes (Red and Green are next to 
>Blue with respect to the enormous number of very different proteins they 
>bind). 

You don't say what you have in your loading buffer.  If you have NAD
present, that could be your problem, since the dye is supposed to bind to
the NAD-binding site.  Just a thought.
Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton,  Alberta     T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca



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