hello Dipankar, Your queries have generated much interest. It would be helpful if you told us which clan the cysteine peptidase you are working with is from. Also it would be very helpful if you could show us the electron density of the "cleaved" peptide in the active site. One presumes that you have a product complex with your peptidase and as has been pointed out this is very interesting. Serine peptidases often have products bound in the S1 to S4 subsites of the enzyme. They remain bound throughout the isolation, purification and crystallization steps of the structure determination.
Regards, Michael James On Mon, Apr 20, 2015 at 2:47 PM, Dipankar Manna <dipankar.biot...@gmail.com> wrote: > 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 < > ponnusam...@googlemail.com> 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 < >> dipankar.biot...@gmail.com> 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 < >>> baerbel.bl...@uni-tuebingen.de> 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 <dipankar.biot...@gmail.com>: >>>> >>>> >>>> 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 <dbon...@ihv.umaryland.edu> >>>>> 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: raj...@itqb.unl.pt >> > > > > -- > Dipankar Manna > Research Scholar > Department of Chemistry > University of Oslo > Oslo, Norway >
