James One thing you should be aware of is that bugs growing in the lysozyme stock solution can have a dramatic effect on the number and size of crystals.
(Way, way) back in 1993 when I was in David Blow's lab we noticed an "ageing" effect with the lysozyme stock solution; if we dissolved freeze-dried lysozyme and set up experiments immediately we got a few large crystals. If, however, we did exactly the same thing except that we kept the lysozyme protein stock overnight in an Eppendorf tube (using a certain batch of tubes), then set up experiments the next morning, we grew dozens of small crystals. The effect seemed to be caused by fungi that were growing in the tubes because fungicides eliminated the effect whereas antibiotics didn't, see below. I have often wondered whether people designing e.g. microgravity experiments that are stored for several days before launching are aware of this! Best wishes Patrick See http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2142306/ Abstract This work investigates the influence of storage of lysozyme in solution on its crystallization. The crystallization of hen egg-white lysozyme exhibits a storage effect (aging) that depends on the length of time the lysozyme solution is stored, after dissolving from freeze-dried powder, before being brought to crystallization conditions. The number of crystals obtained increases, while their size decreases, as the solution ages. Observations suggest that this effect is due to the presence of fungi that multiply in the stored protein solution. This aging effect was used to control nucleation and determine the number and size of lysozyme crystals to be formed in a given sample. Summary of results 1. Under the particular conditionosf these experiments, growth of lysozyme crystals depends on thep resence of a nucleating agent. 2. Aging of lysozyme in solution gives rise to enhanced nucleation. 3. The aging effect is independent of the source of the lysozyme powder. 4. Filtration or centrifugation prior to aging does not eliminate the aging but lessens the number of crystals grown. 5. The agent responsible for the enhanced nucleation can be removed by centrifugation or filtration even after aging has occurred. 6. Desalting increases aging. 7. Aging is prevented by antifungal agents but is not affected by either class of antibiotics. 8. Aging is eliminated when a filtered solution is stored in a sterile vial. 9. Nucleation (and consequently the number of crystals grown) can be controlled by altering the ratio of freshly filtered and aged protein solution just prior to crystallization. On Tue, Jul 26, 2011 at 6:56 PM, James Holton <jmhol...@lbl.gov> wrote: > Does anyone out there have a protocol of growing HEWL crystals that are > all 50-100 microns wide? I gave this project to a summer student > recently, thinking it would be easy, but it is turning out to be more > difficult than I thought. Keep getting sphereulites instead of small > crystals. Yes, I know you can smash a large lysozyme crystal with a > hammer, but that is not exactly what I was going for. What I was hoping > for was a well-defined protocol for growing "reference" crystals that > stay evenly illuminated in our x-ray beams as they rotate. The beam is > 100 um wide. > > I'm sure someone has done this before? > > -James Holton > MAD Scientist > -- patr...@douglas.co.uk Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
