Multiple liquid-liquid partition. II. Theory of Martin--Synge distribution (MSD).
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An apparatus is described that reduces the time required for counter-current distribution in aqueous polymer two-phase systems by a factor of more than five. Phase settling, which has been the time consuming step, is here facilitated by centrifugation. The crucial point in the construction, that allows automatization, is that the transfer step in the counter-current distribution cycle is performed during centrifugation.
The presence of two red cell populations in young chicks has been demonstrated after increasing the settling time used during counter-current distribution with charged 5% Dextran--4% poly(ethylene glycol) two-phase systems. A Fortran program using statistical methods was applied to show the resolution in two peaks (with two or three subpopulations assumed) of the counter-current distribution curves.
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Rat liver plasma membranes, enriched in blood-sinusoidal or bile-canalicular regions by differential and sucrose-gradient centrifugation, were further purified by partitioning in an aqueous polymer two-phase system. This method separates membranes according to differences in surface properties rather than size and density. A several-fold increase in the ratio of leucine aminopeptidase (a bile-canalicular marker) and 5'-nucleotidase to asialo-orosomucoid binding (a blood-sinusoidal marker) was obtained in one fraction, whereas another fraction gave a 2-3-fold increase in ratio of blood-sinusoidal to bile-canalicular markers. Furthermore, the markers for both regions of the plasma membrane, as well as markers for Golgi membranes and lysosomes, showed a heterogeneous behaviour on counter-current distribution.
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