Separation and microgram-scale isolation of sea urchin egg granules by electrophoresis in polyvinylpyrrolidone solution, using horizontal gel electrophoresis apparatus with fluorescence detector.
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.
Chen
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N
Section 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.
Radko
S P
SP
Chrambach
A
A
D000818:Animals; D002021:Buffers; D003594:Cytoplasmic Granules / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007525:Isoelectric Focusing / Q000379:methods; D010063:Ovum / Q000737:chemistry*; D011205:Povidone; D012617:Sea Urchins / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence
D002021:Buffers; D012996:Solutions; D011205:Povidone