The first size separations and the first isolations of subcellular-sized particles by the automated electrophoresis conducted in dilute polymer solutions were achieved. Three components of rat liver microsomes were sequentially isolated by the preparative automated ectrophoresis technique applied in a dilute agarose solution, with a yield of 74 to 85%. By the same technique applied analytically, sea urchin (S. purpuratus) egg cortical granules were shown to exhibit three electrophoretic components. Free-flow electrophoresis is at this time the primary electrophoretic separation method applicable to cells. Although the method remains non-quantitative and relatively rudimentary, it was applied to separating apoptotic from viable human lymphocytes. Complete separation was not achieved, but fractions enriched in either of the two cell types were obtained. A long-term study of membrane protein complexes in """"""""non-denaturing"""""""" detergents was initiated. At present, the analogous studies are universally carried out in the strongly dissociating detergent, SDS, i.e. under conditions that can only detect the dissociated subunits of such complexes, not the biologically active unit itself. """"""""Fusion proteins"""""""" were detected by Western blotting of beta-octylglucoside solubilized sea urchin egg cortical granules. Protein complexes of the immunologically detected """"""""fusion proteins"""""""" were identified by their retardation coefficients. While Ferguson plot analysis provides a gross identification of oligomeric or charge isomeric forms of macromolecules, more specific techniques are needed for separating species with delicate differences in conformation. Such techniques were applied to the separation of conformational isomers of beta-amyloid peptide, a small molecular size marker of Alzheimer's disease. The peptide appears to exist in two conformational states, one of which is beta-sheeted and reacts with Congo Red. Thus, one of the approaches to separating the conformers used high concentrations (to 40%) of polyacrylamide gel in presence of Congo Red. The other used uncrosslinked polyacrylamide solution in CZE a medium previously shown to excel in separating heteroduplex DNA. Both approaches demonstrated four forms of the peptide two of which are size isomeric.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Intramural Research (Z01)
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