strong blue sepharose (?)

[Pascal Mertens]

> 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

You could try to elute with 1mM NADH; to minimize contaminating proteins,
don't change other things (salts, pH) except NADH between washing and
elution buffer.
After your whashings with 2M KCl, there should not be much
non-specifically binded proteins on your column.

Hope this helps

Pascal

-- 
Pascal Mertens
Laboratoire d'Immunologie et Microbiologie
URBM-FUNDP
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5000 Namur, BELGIUM
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