Visualization and Quantitation of Integral Membrane Proteins Using a Plasma Membrane Sheet Assay

Abstract

Preparation of plasma membrane (PM) sheets from intact cells is a simple method for obtaining an immobilized cytoplasmic PM surface that can be probed with antibodies or chemical probes. This method was first reported by Robinson et al. (1) who used it to detect the presence of the GLUT-4 glucose transporter at the PM as a result of insulin stimulation. We have extended this technique in order to use it as a means to quickly and efficiently obtain a highly purified plasma membrane fraction (2). We have successfully used this method to detect the GLUT-4 glucose transporter on the cytoplasmic face of the PM as well as using it to quantify total membrane-associated GLUT-4 by western blotting (2,3). The method can be adapted to measure any strongly associated membrane protein possessing an endofacial epitope (e.g., a protein attached to the PM through a membrane-spanning domain). The method relies on sonic disruption of cells that are coated with a positively charged molecule, which allows the exofacial surface of the PM to be drawn into contact with the solid support on which the cells were cultured. Sonication of these cells results in formation of a “lawn” of adherent membrane fragments oriented with their cytoplasmic surfaces facing away from the surface of cell attachment. This uniform orientation allows access to the cytoplasmic surface of the PM using various probes, after fixation by standard microscopy techniques.