Hi everyone, Many Thanks for your suggestions !! I checked for the everything related to the components i am using : No contamination, not very old, no PEGs etc. And then i followed the way Markov and Prem suggested !! For a trial set up i just et up 10 different concentration variations of my previous conditions (having Tacsimate) and stored at my bench !! I checked after 2 days and cud see some needles back !! But only in 1 out of 10 conditions tested !! The number of needles has also decreased. And the last thing noticeable was that this 1 condition is at lower side (in terms of concentration of precipitant solution) of tested condition as compared to the previous hit solution. So i was wondering is this 1 hit condition (although with poor needles) is just a stochastic hit or should i screen the whole range of precipitant solution again ?? Secondly any suggestion to increase the number of wells having crystals ?? Or the quality of needles ?? Actually i am little concerned with the reproducibility issue also because as i proceed for the optimization, i get confused that is it because of batch variation i m not getting crystals or the tried approach didn't work out ?? Its been long time that i am optimizing these crystals so any suggestions on this matter is highly appreciated !!
Thanks Monica On Mon, Jan 26, 2015 at 10:08 PM, Emilia C. Arturo (Emily) <[email protected] > wrote: > On Mon, Jan 26, 2015 at 8:22 PM, Monica Mittal <[email protected]> >> wrote: >> >>> Hi everyone, >>> >>> I need an advice on some strange thing happening to one of the protein i >>> am working on. I used to purify it and set up trays and get some needle >>> shaped crystals and trying seeding and other methods to optimise them. But >>> recently, it stopped giving crystals even small needles. I am still >>> following the same protocol with same buffer stocks. >>> >> > By 'buffer stocks', do you also mean your stock of precipitating reagent? > It's happened to me that a hit I pursued and optimized vanished when I > opened a new bottle of PEG. It took too long to realize that the old PEG > could have been at a higher concentration than I'd assumed, given its > shelf-age. I increased the PEG % and got back the crystals. > > Good luck, > Emily. > > > And not just once but since last three times it is happening. The purified >>> protein in gel filtration is perfectly fine eluting at same position with >>> symmetrical distribution. However when i am setting up trays under previous >>> conditions, i am not getting the crystals. Instead the drops are quite >>> clear. So i increased the concentration of the protein also from 8 to >>> 11mg/ml, but still the same. Infact i tried adding ligand also but again no >>> crystals. So i would be really grateful if anyone can give a valuable >>> suggestion regarding this problem !! >>> >>> Thanks >>> BR >>> Monica >>> >> >> >
