strong blue sepharose (?)
hgtsei at med-rz.uni-sb.de
Fri Jun 20 02:21:12 EST 1997
In article <33AA441B.678B at comp.kbsi.re.kr>, shkim at COMP.KBSI.RE.KR (Soohyun
> 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
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,
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