Section 4.1.2.3. Dialysis methods

Dialysis also permits ready variation of many parameters that influence the crystallization of macromolecules. Different types of systems can be used, but all follow the same general principle. The macromolecule is separated from a large volume of solvent by a semipermeable membrane that allows the passage of small molecules but prevents that of the macromolecules (Fig. 4.1.2.1b). Equilibration kinetics depend on the membrane molecular-weight exclusion size, the ratio of the concentrations of precipitant inside and outside the macromolecule chamber, the temperature and the geometry of the dialysis cell. The simplest technique uses a dialysis bag (e.g. of inner diameter ∼2 mm), but this usually requires at least 100 µl of macromolecule solution per trial.

Crystallization by dialysis has been adapted to small volumes (10 µl or less per assay) in microdialysis cells made from capillary tubes closed by dialysis membranes or polyacrylamide gel plugs (Zeppenzauer, 1971). Microdialysis devices exist in a variety of forms, some derived from the original Zeppenzauer system (Weber & Goodkin, 1970); another is known as the Cambridge button. With this device, protein solutions are deposited in 10–50 µl depressions in Plexiglas microdialysis buttons, which are then sealed by dialysis membranes fixed by rubber O-rings and subsequently immersed in an exterior solution contained in the wells of Linbro plates (or other vessels). The wells are sealed with glass cover slips and vacuum grease. Another dialysis system using microcapillaries was useful, for example, in the crystallization of an enterotoxin from Escherichia coli (Pronk et al., 1985). In the double dialysis procedure, the equilibration rate is stringently reduced, thereby improving the method as a means of optimizing crystallization conditions (Thomas et al., 1989). Equilibration rates can be manipulated by choosing appropriate membrane molecular-weight exclusion limits, distances between dialysis membranes, or relative volumes.