strong blue sepharose (?)

[Thomas Seib]

In article <33AA441B.678B at comp.kbsi.re.kr>, shkim at COMP.KBSI.RE.KR (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

Hi Soohyun,
you could try including 2 mM ATP in the elution buffer (together with
2 M KCl) for affinity elution. Some proteins bind very strong to dye
matrices, especially when the sepharose is loaded with dye molecules
in high density.
Hope this helps,
Matthias

-- 
Thomas Seib
Kantstr. 12
66292 Riegelsberg