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Preparative electrophoresis in "sieving media" of subcellular-sized particles
1Section on Macromolecular Analysis, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1580, USA. acc@cu.nih.gov
Abstract:
The commercial gel electrophoresis apparatus with intermittent scanning of the migration path and preparative capacity (HPGE-1000, LabIntelligence) is applicable to polymer solutions as well as gels. Unresolved rat liver microsomes can be isolated from 11-15% polyvinylpyrrolidone (PVP) solution by means of a syringe. The automated band isolation technique applied under resolving conditions in dilute polymer solutions allowed for the sequential isolation of three microsome components with 85, 76 and 75% recovery, respectively, under strict control of the dimensions of the volumetric collection module of the HPGE-1000 apparatus. Separations of unlabeled microsomes and sea urchin egg components in dilute polymer solutions have been performed, using detection by "fluorescence reduction". The unlabeled major component of a sea urchin egg homogenate has been isolated from electrophoresis in 1.5% PVP (Mr = 10(6)) solution in 25-50% yield (0.24-4 microg/8 lanes of the HPGE-1000 apparatus). However, since separations of both microsomes and sea urchin egg granules in dilute polymer solutions are restricted to a narrow range of polymer concentrations, their retardation coefficients, KR = d(log mobility)/d(polymer concentration), are not ascertained.