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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 <[log in to unmask]> 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