Figure 1. In (a) the solubility of a typical protein enolase is shown here as a function of ionic strength produced by two different, widely used salts. The regions of the end points of the curves where solubility decreases are called, at low ionic strength, the ‘salting in’ region and, at high ionic strength, the ‘salting out’ region. Both provide opportunities for the creation of supersaturated macromolecular solutions and crystal growth. In (b) the solubility is shown of two typical proteins, hen egg albumin and haemoglobin, as a function of pH. All other parameters are otherwise constant. Both proteins show dramatic decreases in their solubilities at characteristic pH values, a feature that can be used to advantage in creating supersaturated solutions of the proteins. (From Green (1931) Journal of Biological Chemistry 93: 495.)

Figure 2. Drawing showing the principle of free interface, or liquid–liquid, diffusion. (a) Initially an interface is established between a protein solution and a precipitant solution. (b) Over time, diffusive mixing occurs so that regions of the protein solution in the neighbourhood of the interface become supersaturated from precipitant penetration and crystallization is induced.

Figure 3. A drawing showing the process of equilibration through use of microdialysis buttons. Initially the macromolecule samples contain little or no precipitant, but the precipitant concentration rises asymptotically as it penetrates the dialysis membrane. Ultimately, the precipitant concentration is virtually the same on both sides of the membrane, but the protein concentration inside the cavity of the button remains unchanged.

Figure 4. Schematic diagram showing the process of equilibration, through the vapour phase, of a macromolecule containing droplet with a reservoir that is several orders of magnitude larger in volume. The concentration of precipitant is initially higher than that in the droplet, usually about twice, and the two reach a balance when the osmolarity of the drop, through water loss, becomes equal to that of the reservoir. The drop decreases in volume during the process, so that the concentrations of all components, including that of the macromolecule, rise significantly.

Figure 5. The standard configuration for hanging drop protein crystallization experiments is shown here. A tissue culture plate provides 24 wells for reservoirs of about 0.5 to 0.75 mL. Each well can be covered by a cover slip with a drop of protein solution hanging from its underside. The drop of 2 to 20 L equilibrates with the reservoir solution over time through the vapour phase, causing precipitant concentrations to increase in the drop, and the induction of crystallization.