Why not just dialyse the pooled batches into 20mM Tris, 0.5M NaCl
, pH 7.4 or a buffer that is compatible with your enzyme, and after
dialysis, let the pool recirculate on your ConA column overnight or some
hours (depends on the size of your column). Elution will happen in a
much smaller volume, so you may get from 200 ml pooled batch to 1½-2 ml
eluted protein in two easy steps. Only problem is your requirement for
concentration at an earlier step.
Regards
Thomas Krag
Phil Harrison skrev:
>> Hello all.
>> I have been losing my enzyme activity when I concentrate the
> enzyme and could use some help.
> I am purifying a fructosyltransferase from plant tissue. After
> ammonium sulfate fractionation and
> desalting, I have been pooling 4-5 batches and concentrating by
> an Amicon thin channel device.
> These are not sold any more, so let me explain that they are
> like a stirred cell, except the
> enzyme solution is pumped through a thin channel tangentially
> over the surface of the membrane.
> Nitrogen from a compressed nitrogen tank pushes the buffer
> through the membrane while the pump
> recirculates the enzyme solution over the membrane.
>> I am concentrating ca. 200 ml down to ca. 45 ml. Occasionally I
> get ca. 90% recovery of the
> enzyme activity. More often I get 20-25% recovery, and
> sometimes even less.
>> Can anyone suggest a method or methods for concentrating that
> would be more gentle on my
> enzyme? It is a glycoprotein of MW ca. 60 kD. I use a ConA
> column later in the procedure,
> but need concentrating steps earlier.
>> Thanks in advance.
>> Phil
--
********************************************************************
Thomas Krag, M.Sc Phone/work: +45 43232470
Dept. of Clinical Biochemistry Fax/work: +45 43233929
Glostrup Hospital Email/work: tk at dcb-glostrup.dk
DK-2600 Glostrup Email/home: krag at post6.tele.dk
Denmark
********************************************************************
