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.
Currently, low picomole quantities of protein can be subjected to sequence analysis and future improvements in protein sequence analysis technology are likely to allow direct analysis of femtomole quantities of proteins. While these capabilities greatly enhance our ability to characterize proteins, improved sequencing sensitivity will be of limited use without the ability to identify and manipulate nanogram quantities of protein without significant losses. This is a particular problem when source material is available only in limited amounts. One solution to the problem of handling small quantities of protein is to leave the protein of interest in a partially purified state prior to final preparation for protein sequence analysis. A major advance in this direction is the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for final purification. This technique can be readily combined with electroblotting of proteins to membranes for subsequent sequence analysis [l-31. There are, however, two major drawbacks to this approach: (i) in orderto achieve efficient stacking of proteins in the gel there is a limit to the volume of sample (and hence amount of protein) applied to the SDSpolyacrylamide gel; and (ii) most proteins do not renature following SDS treatment, which precludes assaying for their biological activity, thus preventing identification of the protein of interest. We have therefore attempted to combine an existing high-resolution, nondenaturing technique with SDS-PAGE to obtain a two-dimensional method that will allow identification of proteins based on their native biological activity. This technique should be amenable to use following preliminary purification using conventional chromatography procedures and consequently it should be compatible with the preceding step in the purification protocol. Finally, this technique should also allow subsequent preparative scale purification in order to perform amino acid sequence determination of the previously identified protein. We were therefore interested in the technique of immobilized pH gradient isoelectric focusing