Affinity Chromatography

[Jan van der Lee]

Sham,

I have had my problems as well using Sepharose columns.
My solution : add some ethanol or glycerol to your buffer system (5-15%
is usually sufficient) to prevent binding to the Sepharose.
For most (not all) of my separations this worked.

Good luck.

Jan

Sham Nair wrote:

> Hi Netters, I am trying to purify a calcium-dependent lectin
> from the serum of an invertebrate.  After initial affinity
> chromatography (using N-acetyl-D-galactosamine coupled to
> agarose and eluting with EDTA), I tried to separate the
> mixture of proteins in the eluate by size exclusion
> chromatography.  The problem was that I lost the lectin during
> this latter process.  I presumed that the lectin was binding
> to the chromatography media (Sepharose) and tried to carry out
> the separation with EDTA in my lectin suspension, but this did
> not help.  Any suggestions?  Thanks in advance.
>
> Regards,
> Sham