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Shankar, I would suggest to also set up a few plates with your protein in HEPES instead of Tris. I once worked for months trying to improve tiny crystals while my protein was in Tris, pH 7.4, to no avail. I got beautiful crystals when I purified my protein in HEPES, pH 7.4, with everything else the same. HTH, Cale On Fri, Jan 4, 2013 at 11:11 AM, Sankaranarayanan Srinivasan < [log in to unmask]> wrote: > Dear all, > > Thank you very much for all your helpful comments. I will try them and > post on the BB my results. > > Best regards > > Shankar > > > On Wed, Jan 2, 2013 at 11:59 AM, Sankaranarayanan Srinivasan < > [log in to unmask]> wrote: > >> Dear all, >> >> A very happy new year to all. >> >> I would appreciate some expert advice on optimizing a crystallization >> condition in which the initial hits were obtained with ethylene glycol as >> the main precipitant. Here is the summary of things tried. >> >> We have a protein, size (31Kda) and the starting protein buffer is 0.1M >> Tris pH7.5, 0.1M NaCl, 10% glycerol. >> The initial crystal hit was obtained from the emerald cryo kit condition >> that has 0.1M imidazole pH 8.0 , 50% (v/v) ethylene glycol. The crystals >> were tiny (10-20um). A crystallization matrix to obtain better crystals >> by varying the imidazole pH and ethylene glycol concentrations was tried >> from which the best condition obtained was 0.1M imidazole pH 6.5 , 30% >> (v/v) ethylene glycol. The crystals were slightly bigger 50um. >> On trying the additive screen, bigger crystals (200um) were obtained, but >> putting them under the x-ray beam with direct freezing did not yield any >> diffraction spots. >> Trying other cryo-conditions like glycerol and 50-50 paratone/oil mixture >> also yielded similar results. >> Low resolution spots near the beam stop were also not seen. Similarly >> spots indicative of salt was also not seen. It just had hazy ice rings kind >> of stuff. (The beam was definitely on the crystal) >> To check if what we have was salt, a control condition with no protein >> was tried. Also the crystals were run on a gel after thorough washing. Both >> these tests, show that they are definitely protein crystals and not salt. >> Seeding also did not yield any improved crystals. >> I was suggested using di-ethylene glycol, propane diol as alternatives. >> I would greatly appreciate if you can give your opinion on using other >> di-alcohols as precipitants or other ways to improve these crystals. >> I tried searching the PDB to see if someone had actually used ethylene >> glycol as a precipitant, most of them were used as a cryo condition than >> actually as a precipitant. >> >> Thank you very much in advance. >> >> Regards >> Shankar Srinivasan >> >> >