Improved method for the isolation of rat liver plasma membrane.
An improved method for the isolation of plasma membrane from rat liver is presented. Gentle homogenization of perfused livers in buffered isotonic KCL, followed by direct flotation of a low-speed nuclear pellet through a discontinuous sucrose density gradient results in a 32% yield, and 25-fold enrichment for the plasma membrane marker, phosphodiesterase I, in a crude plasma membrane fraction. This fraction contains less than 1% of the mitochondria, and endoplasmic reticulum present in the original homogenate, but is more heavily contaminated with lysosomes and Golgi membrane. Vigorous mechanical disruption of this material, followed by a second discontinuous sucrose density gradient, gives a light plasma membrane fraction with an 80-fold purification and 20% yield of phosphodiesterase I over the original homogete (with further reduction of contaminants).
D000135:Acid Phosphatase / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D005260:Female; D006056:Golgi Apparatus / Q000648:ultrastructure; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure*; D008854:Microscopy, Electron; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis; D051381:Rats
D009251:NADPH-Ferrihemoprotein Reductase; D000135:Acid Phosphatase