Chromatofocusing.

Chromatofocusing is a protein-separation technique that was introduced by Sluyterman and his colleagues between 1977 and 1981 (1-5). Chromatofocusing combines the advantage of high-capacity ion-exchange procedures with the high resolution of isoelectric focusing into a single chromatographic focusing procedure. During chromatofocusing, a weak ion-exchange column of suitable buffering capacity is equilibrated with a buffer that defines the upper pH of the separation pH gradient to follow. A second "focusing" buffer is then applied to elute bound proteins, roughly in order of their isoelectric (PI) points, The pH of the focusing buffer is adjusted to a pH that defines the lower limit of the pH gradient. The pH gradient is formed internally during isocratic elution with a single focusing buffer; no external gradient forming device is required. The pH gradient is formed as the eluting buffer (i.e., focusing buffer) titrates the buffering groups on the ion exchanger. Peak widths in the range of 0.05 pH unit and samples containing several hundred milligrams of protein can be processed in a single step. Chromatofocusing is therefore a powerful analytical probe of protein surface charge, as well as an effective preparative technique for protein isolation. The application of chromatofocusing to silica-based stationary phases for use in a high-performance mode (6) has extended the utility of this technique.