Plasma-Membrane Sheets from Pea Protoplasts

Abstract
Procedures are described for the isolation of stable protoplasts from the apical regions of etiolated peas and for the preparation of plasma-membrane sheets from these protoplasts. Positively-charged silica microbeads were bound to the negatively-charged surfaces of intact protoplasts, exposed surfaces of the microbeads were neutralized and the protoplasts were lysed in a hypotonic buffer. Bead: plasma-membrane sheets pelleted at low centrifugation speeds were purified by repeated washing. Electron microscopy and marker enzyme assays indicated that these membrane sheets were essentially free of contamination by intracellular components and retained plasma-membrane markers at relatively high specific activities.