Advisory Panel Jonathan W . Amy Jack W . Frazer G. Phillip Hicks
INSTRUMENTATION Donald R. Johnson Charles E. Klopfenstein Marvin Margoshes
Harry L. Pardue Ralph E. Thiers William F. Ulrich
Electrofocusing in Gels DANIEL WELLNER Department of Biochemistry, Cornell University Medical College New York, Ν. Υ. 10021
Gel electrofocusing provides the enzymologist, the protein chemist, the geneticist, the immunologist, and the clinician with a versatile supplementary research tool for the analysis and characterization of proteins and peptides. Its simplicity, its high resolving power, and its ease of combination with other techniques indicate that the number of applications to which it will be put will greatly increase in the near future ' " T H E TECHNIQUE of gel electrofocus
ing is one of the most promising new methods for the analysis and char acterization of enzymes, hormones, and other ampholytes of biological interest. It is equal or superior in sensitivity, resolution, and simplicity to other ana lytical methods commonly employed for the study of proteins. Further more, because it separates compounds on the basis of differences in isoelectric point, it provides additional informa tion not readily obtainable by other methods. Therefore, while electro focusing is not likely to displace such methods as electrophoresis, chroma tography, or ultracentrifugation, it represents a valuable supplementary research tool. In this article, the prin ciple of the method will be outlined briefly, the experimental technique will be described, and some examples of its use will be given.
Principle of Method
In an electric field, proteins and other ampholytes move toward the cathode in solutions more acidic than their isoelectric point and toward the anode in solutions more basic than their isoelectric point. At their isoelectric point (when pH = p i ) they carry no net charge and therefore do not mi grate. When a protein is added to a solution containing a pH gradient, and when an electric potential is applied to such a solution, with the anode on the acidic side and the cathode on the basic side, the protein molecules mi grate toward that region of the gra dient where the p H equals their iso electric point. If the pH gradient were stable and if convection and dif fusion were absent, the protein would become concentrated in a sharp sta tionary zone, its position depending on the isoelectric point of the pro tein. An early attempt to exploit this principle for the separation and con centration of proteins was made by Kolin (Î) in 1954. He used a gradient formed between two buffers of different pH and, at the same time, a sucrose density gradient to prevent convective remixing of the separated proteins. Although good separations were achieved in that system, the sharpness of the focusing was limited by the migration of the buffer ions in the electric field and the consequent instability of the pH gradient. This problem was overcome by Svensson (2) and Vesterberg and Svensson (3) by the use of a "natural" pH gradient—i.e., a gradient formed under the influence of an electric current and stable in the presence of the current. This was accomplished by using "carrier ampholytes" consisting of a mixture of many compounds of low molecular weight (
