Thank you Rajesh, That could be a possibility though. I am planning to do some MS analysis from the extra gel bands, if I get any by running SDS-PAGE on the purified protein.
Best, Dipankar On Mon, Apr 20, 2015 at 10:42 PM, rajesh ponnusamy < [email protected]> wrote: > just to add up: Thought about any E.Coli protease as an impurity.. very > small quantity? > > Cheers, > Rajesh. > > > On Mon, Apr 20, 2015 at 9:14 PM, Dipankar Manna < > [email protected]> wrote: > >> 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 >> > > > > -- > Dr. Rajesh Ponnusamy > Macromolecular Crystallography Unit > Instituto de Tecnologia Química e Biológica - ITQB-UNL > Oeiras - Portugal > > phone: (+351) 21 446 96 63 > fax: (+351) 21 443 36 44 > email: [email protected] > -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
