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Separation and microgram‐scale isolation of sea urchin egg granules by electrophoresis in polyvinylpyrrolidone solution, using horizontal gel electrophoresis apparatus with fluorescence detector
Author(s) -
Chen g,
Radko Sergey P.,
Chrambach Andreas
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150190721
Subject(s) - chromatography , polyvinylpyrrolidone , electrophoresis , fluorescence , isolation (microbiology) , chemistry , gel electrophoresis , biology , biochemistry , optics , microbiology and biotechnology , physics , organic chemistry
A homogenate of sea urchin ( Lytechinus pictus ) eggs rich in exocytotic membrane vesicles (granules) was subjected to analytical and preparative electrophoresis in the commercial automated horizontal gel electrophoresis apparatus (HPGE‐1000, LabIntelligence, Belmont, CA) capable of intermittent scanning of the migration path, using buffered solutions of polyvinylpyrrolidone (PVP). The nonfluorescent granules were detected by “fluorescence reduction”, i.e. , a decrease of fluorescence intensity due to the absorbance and/or light scattering properties of the particle. Granules migrated at linear migration rates in buffers ranging from 0 to 2.5% PVP. Two bands were observed and optimally separated in 1.5% PVP solution. As shown by sodium dodecyl sulfate (SDS)‐polypeptide patterns, the material recovered from the bands was qualitatively indistinguishable from the two major fractions A and C of granules previously separated by free‐flow electrophoresis in the absence of polymer. Ferguson plot analysis failed to provide the sizes of the granules in view of the narrow PVP concentration range available for mobility measurement and the unavailability of chemically homogeneous size standards.