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 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [email protected] **************************************************************** On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri <[email protected]> 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
