Rapid, high-yield purification of cell surface membrane using colloidal magnetite coated with polyvinylamine: sedimentation versus magnetic isolation.
A new technique for the magnetic isolation of external plasma membrane from Dictyostelium discoideum is described and compared to a previously published procedure employing sedimentation of silica-coated plasma membrane. The magnetic isolation technique involves coating intact cells with a polyvinylamine-magnetite colloid and overcoating with polyacrylate to form a dense pellicle. The magnetite pellicle totally coated the cells and was not internalized. Coated cells were lysed and membrane fragments retrieved from the cell homogenate using a diverging field electromagnet. The membrane obtained in such a manner was analyzed for marker enzyme activity and cell surface label. The plasma membrane was obtained in high yield (42%) with an average purification of 8-fold. The polyvinylamine-magnetite pellicle shielded the external plasma membrane face to proteolysis by papain and pronase. It also acted as a barrier to alpha-methylmannoside in concanavalin A-carbohydrate competition studies.
D001665:Binding Sites; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D003102:Colloids; D004023:Dictyostelium / Q000648:ultrastructure; D052203:Ferrosoferric Oxide; D007501:Iron; D008280:Magnetics; D008565:Membrane Proteins / Q000032:analysis; D008855:Microscopy, Electron, Scanning; D010087:Oxides; D011145:Polyvinyls; D013499:Surface Properties
D003102:Colloids; D008565:Membrane Proteins; D010087:Oxides; D011145:Polyvinyls; D007501:Iron; D052203:Ferrosoferric Oxide