Dipankar, It is possible that the peptide was cleaved by other proteases present in the soaking solution as impurities. The crystallized protein simply selected that fragment that binds the best. You can try soaking with a lower amount of the peptide (use a smaller drop and concentration).
Alex On Apr 20, 2015, at 1:14 PM, Dipankar Manna 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<mailto: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<mailto: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<mailto: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<tel:%2B49%2070%2071%2029%2075%20359> http://www.ifib.uni-tuebingen.de/research/blaum.html -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
