In article <3E88BF29.1090503 at usfca.edu>, chihara at usfca.edu (USF) wrote:
>We are trying to purify DPPIV from drosophila using standard techniques
>from the lit. We are finding that after putting the homogenate of
>Membrane fraction through a DEAE column, and concentrating we lose all
>the enzymatic activity. We were able to concentrate after gel
>filtration, and there is activity in the fractions off the DEAE column.
> Does anyone have any suggestions as to where to look for the problem?
Maybe, but you don't really describe the problem in an easily
understandable way. What is DPPIV? What is "membrane fraction"
(there are 1001 possible definitions)? What activity is measured
and how is DEAE column(s) run? How do you concentrate the
fractions?
A standard explanation for the sort of thing you describe is an
activity that depends on two separate entities that get resolved
during chromatographic step(s). One thing to try is to mix
every fraction with every other fraction and test the activity
hoping for a clear sign of synergy. OTOH, it is entirely possible
your protein/activity is not stable in dilute/purified form.
Purifying activity by functional assay is a hardest way to learn
living anyway :-) Good luck!
DK
