Dear Barbel, Thank you!
Yes you are right that I did the SDS-PAGE with bigger substrate. Regarding peptide, we did check the MS and HPLC profile for the peptides which clearly shows that there should not be any cleaved peptides! Best, Dipankar On Mon, Apr 20, 2015 at 9:46 PM, Bärbel Blaum < [email protected]> wrote: > 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 > -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
