I suppose you do the SDS PAGE test not with the peptide but some
bigger substrate. Are you sure your peptide is intact *before*
soaking? I.e. have you checked the batch yourself with MS or NMR? We
regularly get small compounds (sugars) that turn out not to be what
the label says.
Bärbel
Zitat von Dipankar Manna <[email protected]>:
Dear Bonsor,
Thanks for your suggestions!
It need 2-3 weeks to get the fully grown crystals. And harvest, freeze and
data collection just take usually next 3-5 days. I usually incubated
substrate overnight. Initially I was purifying with the same column as the
WT but in the next batch I used new beads to purify the mutant as you
categorically pointed out. But results are the same, I mean I got the same
cleaved peptide density! I tried soaking with different time frames and
with different peptide concentrations as well but in this case I can't see
any peptide density at all.
Best,
Dipankar
On Mon, Apr 20, 2015 at 9:06 PM, D Bonsor <[email protected]> wrote:
First of all, you don't say how long it took to first set up crystals, for
them to grow, harvest, freeze and collect data on. Secondly how long did
leave the peptide/substrate for your SDS PAGE experiment? If they are of a
different time scale e.g. 6 hours v.s. 30 days, it may be that your enzyme
is not totally dead.
Also how did you purify the alanine mutant? If you purified it on the same
columns/beads as the WT protein you may have a residual amount of active
protein which could cleave your peptide over the course of crystallization.
You may want to use fresh beads, or treat columns with pepsin or sodium
hydroxide.
Not real answers I am afraid, more like suggestions.
--
Dipankar Manna
Research Scholar
Department of Chemistry
University of Oslo
Oslo, Norway
--
Bärbel Blaum, Ph.D.
Interfakultäres Institut für Biochemie (IFIB)
Hoppe-Seyler-Strasse 4
D-72076 Tübingen
Germany
+49 70 71 29 75 359
http://www.ifib.uni-tuebingen.de/research/blaum.html
