Development of high-performance techniques for the separation and analysis of minor protein components in human plasma

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In the course to analyze total proteins in human plasma and lymphocytes, we have used two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) since it has the best resolution for the analysis of proteins. However, most of the workers on total protein analysis are using 2-D PAGE in denaturing conditions, to obtain structural information of the polypeptides separated on the 2-D gels. However, ultimate aim of protein analysis must be the re-construction of protein functions in living cells or organs. For this purpose, we have been using 2-D PAGE in the absence of denaturants. In this research project, we proposed a combination of four 2-D PAGE techniques (Type-I, Type-II, Type-III and Type-IV) to correlate structural information of polypeptides (obtained by Type-IV 2-D PAGE) with information on protein functions (obtained by Type-I 2-D PAGE).Further, we developed new electrophoretic techniques of protein separation and recovery which overcome the shortage of conventional 2-D PAGE te … More chniques. In conventional 2-D PAGE, after electrophoresis proteins are fixed on the slab gel, stained with a dye or silver nitrate, quantitated by an image analysis software installed in a microcomputer, and extracted from the gel piece for further structural analysis. Most of these procedures require manual operation and their automation is almost impossible. During 1996-1998, we have developed techniques of capillary electrophoresis for the separation of proteins. Capillary electrophoresis is characterized by on-column detection, on-column quantitation, and full-automation of the separation process. We established the conditions of capillary isoelectric focusing and capillary SDS electrophoresis and applied them for the separation of human plasma proteins. Then we extended these conditions of capillary electrophoresis aiming to establish high-performance techniques for protein separation and recovery. We have constructed several apparatus and devises for this purpose and we are evaluating their performance in the separation of minor protein components in human plasma. Less