Purification of plasma membranes by aqueous two-phase affinity partitioning.
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.
Persson
A
A
Chemical Center, University of Lund, Sweden.
Jergil
B
B
D000818:Animals; D015415:Biomarkers; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D002498:Centrifugation; D003911:Dextrans; D008099:Liver / Q000648:ultrastructure; D008297:Male; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D014909:Wheat Germ Agglutinins
D015415:Biomarkers; D003911:Dextrans; D014909:Wheat Germ Agglutinins; D011092:Polyethylene Glycols