Preparation of a highly purified surface membrane fraction from rabbit polymorphonuclear leucocytes by high-voltage free-flow electrophoresis.
A surface membrane fraction of high purity and good yield has been prepared from homogenates of rabbit peritoneal polymorphonuclear leucocytes, using a preliminary sorbitol density gradient sedimentation followed by preparative high voltage electrophoresis in a thin flowing buffer film. Enrichment values for the plasma membrane marker enzyme 5'-nucleotidase and 125I-labelled Lens culinaris lectin, after the latter had been applied at the whole cell level, were 18-fold and 6-fold, respectively. Contamination of the surface membrane fraction by other organelles was negligible and approximately 1 mg of surface membrane protein can be obtained from 2 . 10(9) leucocytes. A triacylglycerol-rich, protein-poor fraction that lacks any definable structure in electron microscopy separates discretely from the surface membrane vesicles during electrophoresis. It is considered that this may be a contaminant not previously recognized as present in membrane fractions prepared by more conventional procedures.
D015720:5'-Nucleotidase; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D004586:Electrophoresis / Q000379:methods; D006867:Hydrolases / Q000097:blood; D007770:L-Lactate Dehydrogenase / Q000097:blood; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification; D008854:Microscopy, Electron; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology / Q000648:ultrastructure*; D009708:Nucleotidases / Q000097:blood; D011817:Rabbits
D008565:Membrane Proteins; D007770:L-Lactate Dehydrogenase; D006867:Hydrolases; D009708:Nucleotidases; D015720:5'-Nucleotidase