The dynamic nature of insulin-sensitive glucose transporter isoform 4 (GLUT4) storage vesicles (GSVs) makes their characterization challenging. Fractionation techniques can facilitate isolation of GSVs from insulin-sensitive cells. In this protocol, we describe preparation of a total membrane fraction from 3T3-L1 adipocytes. The resulting pellet contains all membranes and allows for easier identification of membrane proteins, including the insulin-sensitive pool of GLUT4. A method for concentration of the soluble fraction is also included.
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