help: enzyme purification

Jennifer Humphries 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.

Jennifer





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