Imporved method for isolation of plasma membrane on cationic beads. Membranes from Dictyostelium discoideum.
The plasma membrane from Dictyostelium discoideum was routinely purified 35-fold by an improved technique using beads coated with positively charged polymers. Cells were attached to the beads and bare regions between the cells were neutralized with a polyanion. The neutralization decreased contamination of the bare regions by intracellular proteins released when cells were disrupted to leave behind beads coated by plasma membrane. The neutralization increased the purification as measured by membrane-bound 125I-labeled concanavalin A. Contamination by markers for various intracellular components was markedly decreased. Various bare-site neutralization reagents were evaluated and gave different results depending upon their charge density and molecular weight. The pH of the neutralization was critical. The optimum pH for cell attachment to beads, 5.0, had little effect as regard bare-site neutralization. A new procedure is given that optimizes the essential features for the plasma membrane isolation on beads.
D002411:Cation Exchange Resins; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000648:ultrastructure*; D003208:Concanavalin A; D004023:Dictyostelium / Q000648:ultrastructure*; D005656:Fungal Proteins / Q000032:analysis; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis; D008855:Microscopy, Electron, Scanning; D010455:Peptides
D002411:Cation Exchange Resins; D005656:Fungal Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D010455:Peptides; D003208:Concanavalin A