Protein growth techniques
There are four major techniques used to grow protein crystals: batch crystallization, vapour diffusion, liquid-liquid diffusion and dialysis. The typical time for protein crystal growth is 1 to 3 weeks, although times can vary from several hours to a year. Batch crystallization is the oldest and simplest method: the protein to be crystallized is mixed with the crystallizing agents at the required concentrations at the start of the experiment and left to crystallize.
The various diffusion/dialysis methods are dynamic systems that involve attaining an equilibrium between an aqueous drop containing the protein solution and a reservoir containing the crystallizing agents (i.e. buffer, precipitants, additives). A gradual change of the conditions takes place in the drop en route to equilibrium, often resulting in the formation of crystals.
No one method is considered superior to another. The choice of method depends on the protein involved, its quantity and mainly on the preference of the experimenter. Vapour diffusion and batch are the most widely used techniques mainly due to the ease with which they can be set up in comparison with the other methods. Moreover, automation has been developed for both.
Another approach is to employ microgravity conditions to eliminate convection and sedimentation. This allows solely diffusion-driven conditions to be established. These are thought to be ideal for high-quality crystal growth and, in particular, the growth of crystals with weak intermolecular forces and lattice interactions. Microgravity conditions for crystallization can be partially mimicked by growing crystals in oil.
This method, pioneered at Imperial College, involves "containerless" growth with the drop containing the protein and the crystallizing agents suspended between two different oils with carefully chosen densities (see figure). The bottom layer is a high-density (1.27 g cm-3) fluorinated silicone fluid and the top layer contains low-density standard silicone fluid (0.92 g cm-3).