I assume you were using vapour diffusion. In that case, you could also try microbatch to cover the possibility of the plate slowly drying out and concentrations increasing beyond those of the original screen. Dmitry
On Fri, Oct 31, 2014 at 12:07 PM, R. M. Garavito <rmgarav...@gmail.com> wrote: > Although three months is a long time, it is no completely unheard of, and > does not require the invocation of proteolysis. The longest time I have > heard of is ~1 yr, so count yourself lucky. However to get good advice, as > well as to use it, you need to ask yourself several questions: > > 1. What kind of crystals are they? Protein, salt, etc? If they are salt, > don't pursue this condition. > > 2. How many crystals did you get? One or 2 in a drop or a microcrystal > shower. And of what kind? Single, well shaped, rosettes, needle clusters, > or something that looks crystalline. Screen more broadly around your > initial hit. > > 3. How many times have your tried to repeat this? Once, twice, or more? > Did you try setups in duplicate? If so, did you get reproducible results? > Have you actually screened around these conditions, varying each component > systematically (PEG, salt, pH, buffer, etc.)? > > 4. What method did you use? And in what kind of container? For one > thing, we don't completely trust the integrity of our setups for longer > than 2 months. All containers leak water slowly, so when crystals take > longer than 2 months to grow (a) the real conditions are at much higher > values than you naively think (i.e., the drop has dried out more than you > expected) or (b) other components are crystallizing, for example a zinc > salt. It depends what else is in your protein buffer, as well. > > To quicken protein crystallization (which is not always a good thing), > increase your protein concentration (by 1.5-2x) and/or PEG concentration > (such as screening up to 40% PEGmme 550). Sadly, crystallization is a > combination of thermodynamic and kinetic factors: you can get crystals > (sometimes a single crystal only) when just outside the truly optimal > conditions, but this may be only a sporadic event. You got to keep > screening. > > Good luck, > > Michael > > > ****************************************************************** > *R. Michael Garavito, Ph.D.* > *Professor of Biochemistry & Molecular Biology* > *603 Wilson Rd., Rm. 513* > *Michigan State University * > *East Lansing, MI 48824-1319* > *Office:* *(517) 355-9724 <%28517%29%20355-9724> Lab: (517) > 353-9125 <%28517%29%20353-9125>* > *FAX: (517) 353-9334 <%28517%29%20353-9334> > Email: rmgarav...@gmail.com <garav...@gmail.com>* > ****************************************************************** > > > > > On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri < > vijaypkuma...@gmail.com> wrote: > > Dear all, > > I am trying to crystallize a 30 kD protein. Protein crystals are formed > after 3 months. The crystals are formed in the following condition: > 0.01M Zinc sulphate > 0.1M MES monohydrate pH 6.5 > 25% v/v PEG monomethyl ether 550 > > Please suggest me how to grow these crystals faster. > > Thanking you > > -- > Vijaykumar Pillalamarri, > UGC-JRF, > C/O: Dr. Anthony Addlagatta, > Senior Scientist, > CSIR-IICT, Tarnaka, > Hyderabad, India-500007 > Mobile: +918886922975 > > >
