Microbatch Crystallization
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Microbatch Crystallization

Oryx and IMPAX was originally developed to carry out protein crystallization by the microbatch method by Douglas Instruments in collaboration with Imperial College, London. The method was developed to allow theoretical studies but it is now generally used for routine crystallization, since it is very rapid and uses only about 0.2 - 1l of protein (0.1 to 1l for Oryx 6) per trial.

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 l. Droplets with volumes from 0.4 to 2 l (0.2 to 2l for Oryx 6) are dispensed at the bottom of the wells.

Image of microtip

A special 5 bore microtip, and highly accurate motorized syringes, are used to dispense these small droplets accurately. The dispensing error is around 20 nl.

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" (published in the Journal of Crystal Growth 168 (1996) 170-174). 

Microbatch gives superior crystals for data collection in about 50 % of proteins. See our references for examples.  D'Arcy et al. show photographs of crystals of three research proteins which gave improved crystals with microbatch (using the D'Arcy diffusion technique, see below).  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). 

Microbatch can be used for controlling nucleation by carefully varying the temperature (Blow et al. Protein Science. 3 (1994), pp 1638-1643). 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 vapor diffusion is always tried in the final stages of optimization.

The main disadvantage of microbatch is that it is relatively difficult to change conditions using the same protein sample.

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.

Microbatch screening finds more leads
than VD in a given time
mbvsvd.gif (3919 bytes)
J.Crystal Growth 168(1996), p170-4 or: http://www.douglas.co.uk/rep2.htm

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 (now at Morphochem, Basle) 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." Journal of Crystal Growth 168 (1996) 175-180.

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


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