strong blue sepharose (?)

[Austin Seo]

Soohyun Kim wrote:
> 
> Dear, everyone
> 
>         I am purifying some enzymes requiring NAD with blue sepharose of
> Pharmacia. I used 50 mM phosphate or 20 mM Tris, pH 7.0 as starting
> buffer and above buffer containing 2 M KCL or NaCl as elution buffer. I
> confirmed the enzyme activity before chromatography, but I could not
> find the activity after chromatography. I used three column volumes of
> elution buffer. Where is the enzyme? How can I detach the guys from blue
> sepharose? Please send me your idea.
> With best regards.
> 
> Soohyun Kim
> 
> Korea Basic Science Institute

Use 8-10mM NADH in your final wash buffer (it is a lot). It should come
out within 50ml, depending on the size of your column. Some people have
used a mixture of polyethylene gylcol and 1mM NADH, but this is less
farting around.

Assay fractions for activity (*not* protein), and then pool and dialyse
those with the highest activity (we use ultrafiltration units with 5000
to 10000 MW cutoff).

Good luck.

-- 


Austin Seo (Hae-Jin)



Kinsmen Laboratory of Neurological Sciences

Department of Psychiatry

University of British Columbia

e-mail: austinso at interchange.ubc.ca