Alex Kolesar Micrograph

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. Typically, the cytoplasmic surfaces are probed using an antibody directed against an endofacial epitope of a membrane-bound protein of interest, and this is, in turn, is detected by a flu-

orescent secondary antibody (see Fig. 1). Alternatively, the entire PM fraction can be solubilized and assayed for the total quantity of the protein of interest. Cells to be viewed by microscopy can be grown on coated cover slips, tissue culture microscope chamber slides, or directly on tissue culture dishes (35 mm or larger) if an inverted microscope is to be used for viewing, or on cover slips if an inverted microscope is not used. Cells to be used for PM fractionation are grown on tissue culture dishes of a size sufficient to produce a detectable amount of the protein of interest. For detection of GLUT-4, 35-mm dishes are sufficient to achieve a strong signal by Western blotting.

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Supplements For Diabetics

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