Angela
Rather than make this more complicated than it sounds, be sure that
the ending conditions are not so polar that your protein is still
retained. Typically HIC starts at 1.8M (NH4)2SO4 (though it need
not) and decreases to just the buffer. I may have misread your
conditions, but did you stop at 1M (NH4)2SO4 or start at it?
Dave Schooley's suggestions are good if the protein is unstable to
metals, and Andy Alpert's are good if the protein is so hydrophobic
that it doesn't want to come off the column material you chose.
However, it is soluble, you tested activity before and you had a good
chromatogram of what ever came off, and butyl-sepharose is not that
hydrophobic. So look at the void volume peak to make sure it stuck
and run just buffer just make sure it didn't come off, before you
start with more drastic conditions.
Use of 1-5% EtOH on the butyl-sepharose might be acceptable, but
watch out for bed collapse. Let us know if the activity was recovered
with your subsequent experiments so we can learn too.
Best regards,
Amos Heckendorf
NEW URL: http://www.nestgrp.com please bookmark the change!
>Hi everyone,
>I try to purify an enzyme (bezoate dihydrodiol dehydrogenase) from
>Rhodococcus opacus (Gram +).
>I noticed that the optimal pH for this enzyme is 9, so I started an
>anion exchange chromatography with buffer pH 8 because it was supposed
>that 9 is too high. The chomatogram was nice and i got activity in a
>fraction. The problem came afterwards when I tryed a Hydrofobic
>Interaccion Chrom. I precipitated the protein pool with (NH4)2SO4 and
>resuspended them with puffer pH8+salt. The activity was high so I did a
>HIC (phenyl sepharose) with decreasing concentration of TrisHCl 25mM pH8
>+ (NH4)2SO4 1M. I got a good chromatogram but no activity in any
>fraccion. Then I tryed a butyl Q-sepharose column and also didnĄt work.
>Last time I used a buffer pH 7,5 and I had again no activity.
>Do you have any idea where the problem can be?
>
>Thanks
>
>Angela
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