High-Resolution Localization of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labeling (SDS-FRL)

Abstract

Visualizing molecular localization at high resolution contributes to understanding of their functions and roles in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study high-resolution two-dimensional distribution of transmembrane proteins and their tightly associated proteins on platinum-carbon replica. During treatment with SDS, unfixed proteins and intracellular organelle are dissolved and integral membrane proteins captured and stabilized by carbon and platinum deposition are denatured, retaining most of their antigenicity, and exposed on exoplasmic and protoplasmic surfaces of lipid monolayers. The exposure of these antigens on the surface of replica facilitates the accessibility of antibodies and therefore provides higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples and an additional procedure for fluorescence-guided electron microscopy for replica immunolabeling.