Jennifer Humphries (jlhumphr at taurus.oac.uci.edu) wrote:
: I know that the enzyme will elute with a 400mM NaCl soln because I
: injected the enzyme onto the column with this high salt concentration and
: was able to recover the enzyme. It is only
: when I try to inject the enzyme onto the column at the lower salt
: conc.(150mM) and then increase the concentration to 400mM that I no longer
: recover the enzyme, which doesn't make sense because it should at least be
: coming out at the very end of the gradient with the 400mM salt solution.
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?
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?
Good luck,
Steve Dahl
