Monolayer freeze‐fracture—a modified procedure

Abstract

AbstractMonolayer freeze‐fracture of biological membranes is a valuable tool for integrating membrane morphology with biochemical analysis of membrane components. This correlation has been restricted by the purity of the biochemical sample. In this article, the method is reviewed, and an improved method is described. The essential modification was the use of a polysaccharide‐coated microscope slide, instead of a copper plate, to cover cells attached to a polylysine‐coated coverslip. It was found that proper freeze‐fracture will not occur unless there is a distinct temperature gradient, with its accompanying stresses, across the cell monolayer during the freezing process. This gradient is achieved by using glass slides of different thickness to cover each side of the monolayer. Comparison of the results with those obtained when using a copper‐glass system demonstrated a consistently purer sample for the glass‐glass system, with whole‐cell contamination of the external membrane leaflet being reduced to 0.4%. Problems associated with obtaining pure samples for biochemical analysis are discussed, and the results of freeze‐fracture with the glass‐glass and glass‐copper systems are compared. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of polypeptides associated with the separate halves of the erythrocyte membrane demonstrated that band 3, the anion transport protein, separates with the cytoplasmic face, whereas only sialoglycoproteins and their fragments are retained in the external face. This finding, obtained with the glass‐glass system, is consistent with results of our earlier freeze‐fracture study that used a copper‐glass system which showed that covalent bonds may be broken during this procedure.