Coating cells with colloidal silica for high yield isolation of plasma membrane sheets and identification of transmembrane proteins.
Plasma membrane (PM) can be isolated by binding to a positively charged solid support. Using this concept, we have developed a novel method of PM isolation using cationic colloidal silica. The method is designed for the comparative study of various physiological states of PM and for transbilayer protein mapping. The procedure consists of coating intact cells with a dense pellicle of silica particles and polyanion. Since cells remain intact during pellicle formation, the external face of the PM is selectively coated. The pellicle greatly enhances PM density and stabilizes it against vesiculation or lateral reorientation. Upon cell lysis, large open sheets of PM are rapidly isolated by centrifugation. PM from Dictyostelium discoideum was prepared by this method. Marker enzymes, cell surface labeling and microscopy demonstrate that the PM was isolated in high yield (70-80%) with a 10-17-fold purification and only low levels of cytoplasmic contamination. The pellicle remains intact during cell lysis and membrane isolation, shielding the external surface of the membranes up to 92% from chemical or enzymatic attack. The PM can thus be labeled selectively from inside and/or outside. Transmembrane proteins were identified in Dictyostelium PM by means of lactoperoxidase iodination and autoradiography.
D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000648:ultrastructure*; D004023:Dictyostelium; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D008855:Microscopy, Electron, Scanning; D012822:Silicon Dioxide
D008565:Membrane Proteins; D012822:Silicon Dioxide