Microbatch-Under-Oil Crystallization

The microbatch-under-oil method, and the IMPAX crystallization system, were invented by Imperial College and Douglas Instruments in collaboration (Chayen et al. J. App. Crystallography. 23(1990) 297-302). We wanted a simple miniature method that could be used for theoretical studies, but it soon became clear that the approach was very useful for routine crystallization, including screening, optimization and crystal harvesting.

Like the original batch crystallization methods that were used in the early days of protein crystallization, microbatch involves the simple combination of protein with precipitants, buffers, etc., generally without any subsequent concentration step. The ingredients are simply mixed at their final concentrations.

Because very small volumes are used, the droplets must be covered with paraffin oil to prevent evaporation. The Douglas Vapor Batch Plate is generally used. These have 96 wells, each holding 9 ul. Droplets with volumes from 0.4 to 2 ul (0.2 to 2ul for Oryx 6) are dispensed at the bottom of the wells.

Image of microtip

A special 3-bore (screening etc.) or 7-bore (optimization) microtip, and highly accurate motorized syringes, are used to dispense these small droplets accurately. The dispensing error is around 20 nl.

Microbatch gives superior crystals for data collection in about 50 % of proteins. See our references for examples.  Naomi Chayen and others have successfully improved crystal quality by using microbatch to control nucleation, so that different conditions could be used for nucleation and growth.  In another example Conti et al. found that crystals of firefly luciferase were not stable to temperature changes with vapor diffusion.  This problem was overcome using microbatch (see research report 3).  A final advantage is that the thick skins that often form with vapor diffusion are eliminated (e.g. Pearl et al. EMBO Journal 13. (1994), p 5810).  Microbatch compliments, rather than replaces, vapor diffusion, and we recommend that both methods should be tested.

Microbatch crystals

Microbatch crystals of Carboxypeptidase G2, reverse transcriptase,
and crustacyanin. The light circle is the flat bottom of the well, diameter 1.4 mm.
(Images kindly provided by Prof. Naomi Chayen.)

As shown in the table below, microbatch is more effective than vapor diffusion for screening: for a given amount of time and material, more hits are found.  For more information see research report 2.3 "A Comparison of Microbatch and Vapor Diffusion for Initial Screening of Crystallization Conditions" (J.Crystal Growth 168(1996), p170-4). 

Microbatch screening finds more leads than VD in a given time

mbvsvd.gif (3919 bytes)

J.Crystal Growth 168(1996), p170-4

D'Arcy Diffusion Technique

Some proteins give better crystals with vapor diffusion, presumably because the slow concentration of the droplet during equilibration allows crystallization at low levels of supersaturation of the protein. This slow concentration can be achieved in microbatch by covering the droplets with an oil which allows slow evaporation, thus mimicking vapor diffusion. Allan D'Arcy recommends a 50:50 mixture of silicone oil (Dow Corning 200/1cs) and light paraffin oil (Merck 7174.2500).  For more details see A. D'Arcy, C. Elmore, M. Stihle, J.E. Johnston.  "A novel approach to crystallising proteins under oil." J.Crystal Growth 168(1996), 175-180.

This method is particularly useful for screening - see research report 2.3

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