Improved method for identification of proteins using two-dimensional electrophoresis with immobilized pH gradient...

Recent advances in protein sequence analysis now permit the determination of partial N-terminal and internal primary structure from low picomole quantities of protein. The major remaining hurdles to sequence analysis of small amounts of protein are the identification, isolation, and handling of microgram and submicrogram quantities of protein. The technique of two-dimensional electrophoresis using immobilized pH gradient isoelectric focusing circumvents many of these problems. However, poor correlation between the first and second dimension have prevented use of this technique for the identification of some proteins which can only be assayed prior to the denaturing conditions used in the second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis procedure. An improved method is presented which allows correlation of the native biological activity (first dimension) to a silver stained protein (second dimension) with a high degree of confidence.