Control of the rate of evaporation in protein crystallization by the 'microbatch under oil' method

Abstract

Microbatch crystallization under oil is a powerful procedure for obtaining protein crystals. Using this method, aqueous protein solutions are dispensed under liquid oil, and water evaporates through the layer of oil, with a concomitant increase in the concentrations of both protein and precipitant until the nucleation point is reached. A technique is presented for regulating the rate of water evaporation, which permits fine tuning of the crystallization conditions as well as preventing complete desiccation of the drops in the microbatch crystallization trays.

Figure 1

Douglas Vapor Batch hydrophobic crystallization plate. Crystallization droplets are dispensed under oil into the wells in the central part of the plate. The humidity within the tray above the oil-covered droplets is determined by the vapor pressure of the liquid in the reservoirs.

Figure 2

Image of a crystallization droplet under oil in a well in the crystallization plate. The measured diameter of the droplet is indicated by the double-headed arrow.

Figure 3

Dehydration of crystallization drops. Volumes of crystallization drops as a function of time. Each point represents the average diameter for eight crystallization droplets. Standard deviations of 1σ are typically below 15% of the calculated volumes (not shown).