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Subcellular fractionation of rat liver homogenates using two-polymer phase systems in a toroidal-coil centrifuge.

ABSTRACT: The principal organelles of rat liver homogenates were fractionated by two-phase partition chromatography using toroidal-coil centrifugation with a mixture of dextran T 500 and poly(ethylene glycol) 6000 in 0.26 M-sucrose containing 10 mM-sodium phosphate/phosphoric acid buffer, pH 7.4. The effects of varying the following parameters on organelle elution profiles, as reflected by their marker-enzyme activities, were studied: centrifuge speed; the composition and relative proportion of dextran-rich and poly(ethylene glycol)-rich phases in the eluent; flow rate; sample volume; homogenate concentration; helix diameter; tubing bore and the number of loops in the coil. Optimal resolution of the organelles was achieved with a toroidal coil of internal diameter 1.07 mm with a 4.55 mm helix diameter on a 0.42 m-diameter rotor running at 1000 rev./min. The eluent was prepared by combining, in a ratio of 93:7 (v/v), the poly(ethylene glycol)-rich upper phase and dextran-rich lower phase obtained from a phase mixture containing 3.3% (w/w) dextran and 5.4% (w/w) poly(ethylene glycol). The flow rate of the eluent was 14ml/h. Optimal conditions for separation of the organelles were evaluated. Resolution of plasma membrane and lysosomes was achieved. Separation of endoplasmic reticulum, which showed marked heterogeneity, from plasma membrane was also demonstrated. DNA and marker enzymes for peroxisomes, mitochondria and cytosol showed distinct elution profiles.

SUBMITTER: Heywood-Waddington D

PROVIDER: S-EPMC1153278 | biostudies-other | 1984 Feb

REPOSITORIES: biostudies-other

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Selective and mild fractionation of microalgal proteins and pigments using aqueous two-phase systems.

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| S-EPMC6099415 | biostudies-literature

The dynamics of phase partition. A study of parameters affecting rat liver organelle partitioning in aqueous two-polymer phase systems.

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| S-EPMC1146673 | biostudies-other

Fractionation of rat liver plasma-membrane regions by two-phase partitioning.

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| S-EPMC9439200 | biostudies-literature

Effects of iodination on the distribution of peptide hormones in aqueous two-phase polymer systems.

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Analytical subcellular fractionation of needle-biopsy specimens from human liver.

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2019-02-19 | GSE106094 | GEO

Experiment C, Subcellular fractionation of rat liver

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2016-10-06 | MSV000080232 | MassIVE

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