Purification of plasma membranes by aqueous two-phase affinity partitioning

Anal Biochem. 1992 Jul;204(1):131-6. doi: 10.1016/0003-2697(92)90151-v.

Abstract

A rapid method for purifying rat liver plasma membranes of high purity and yield is described. Squashed liver was homogenized in an aqueous polyethylene glycol-dextran two-phase system. After phase separation and reextraction of the bottom phase with fresh top phase, the combined polyethylene glycol-rich top phases were affinity partitioned in the presence of borate buffer with new bottom phase containing dextran-linked wheat-germ agglutinin. Under these conditions the lectin selectively pulled plasma membranes into the dextran-rich bottom phase, while other membranes preferentially distributed in the top phase. The lectin-containing bottom phase was reextracted with fresh top phase before collecting the purified plasma membranes by centrifugation. This protocol resulted in a preparation that was 30- to 40-fold enriched compared to the homogenate in plasma membrane markers for both the apical and basolateral domains and had yields of 55-70%. The contamination by other membranes was low. The entire procedure was completed within 90 min. The method should be useful for purifying plasma membranes also from other sources.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers
  • Cell Fractionation / methods*
  • Cell Membrane / enzymology
  • Cell Membrane / ultrastructure*
  • Centrifugation
  • Dextrans
  • Liver / ultrastructure
  • Male
  • Polyethylene Glycols
  • Rats
  • Rats, Inbred Strains
  • Wheat Germ Agglutinins

Substances

  • Biomarkers
  • Dextrans
  • Wheat Germ Agglutinins
  • Polyethylene Glycols