ISOELECTRIC-FOCUSING IN IMMOBILIZED PH GRADIENTS

Immobilized pH gradients (IPG), introduced as early as 1982, have been plagued by several problems that have hampered their use over the years. We review here recent technical advances that have led to the solution of these problems and to the development of a successful and trouble-free IPG technique. In particular, It was discovered that the buffers used (acrylamido weak acids and bases) were prone to two major degradation pathways, hydrolysis at the amido bond and autopolymerization. A new generation of these buffers is described, in which both degradation routes are completely inhibited. In addition, the formulas and physicochemical properties of these acrylamido buffers are described for the first time. These buffers consist of a set of eight weak acrylamido acids and bases, with pK values evenly distributed in the 3.1-10.3 pH range, and two strong titrants: 2-acrylamido-2-methylpropanesulfonic acid (pK = 1) and QAE-acrylamide (pK > 12). The advantage and limitations of the IPG technique are evaluated. In particular, examples of difficult separations at very acidic and alkaline pH values, where conventional isoelectric focusing would fail, are described. Applications of the IPG technique to the fields of human and animal genetics, for the resolution of "neutral or electrophoretically silent" mutants, are discussed.