Hi Shankar, I don't claim to be an expert, but I've used ethylene glycol as a cryoprotectant many times, and 30% (v/v) would probably be sufficient for proper freezing without any further addition.
Did you try and shoot a crystal at room temperature? Hth, Dave On Jan 2, 2013 5:59 PM, "Sankaranarayanan Srinivasan" <texs...@gmail.com> 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 > >
