help: enzyme purification
jlhumphr at rigel.oac.uci.edu
Fri Mar 14 18:21:05 EST 1997
> I'm not sure I'd use the elution test described above as a hard and fast
> rule. In solution, yes, you have shown that your enzyme does not bind to
> the column in 400mM salt. This does not necessarily mean, however, that
> once bound to the column, the molecule will release at 400 mM. Binding
> to the resin throws another parameter into the mix. How much higher have
> you taken your elution buffer? 500mM? 1M?
I didn't consider this. I just assumed that if it didn't bind at 400mM
that it would also release at 400mM, which I guess I can't assume. I did
try running a higher salt concentration also, but no luck. The problem is
I got the enzyme to elute previously under the exact same gradient, but
now it doesn't work. The back pressure has also doubled so I think maybe
I clogged up the column somehow and that is affecting the elution pattern.
> Also remember that your elution assay is enzyme activity, so if by some
> major stroke of bad luck your enzyme loses activity after interacting
> with the column matrix it will, um, never elute. Very rare, but not
> unknown. Any chance its purified enough at this step to use a uv monitor?
I have tried using a UV monitor, but there is a huge solvent peak that
interferes because I only have a 206nm filter and the solvent has Cl ions.
Maybe I should invest in a 280nm filter.
Thank you for your suggestions. Wish me luck.
More information about the Proteins