Western Blotting Analysis

The immunodetection of proteins bound to a membrane has widespread applications in plant biochemistry and molecular biology, including the identification and semiquantitative determination of foreign proteins expressed in transgenic plants. The approach is usually applied to protein transferred from electrophoretic separations (see Chapter 34 ), which allows positive identification to be combined with the provision of information about the protein M r charge, pI, and so on. This is commonly called Western blotting, and provides the operator with a wide range of options for choice of membrane type, transfer system, and detection system. We will initially discuss these options, and then provide detailed step-by-step instructions for a well-established method of protein transfer and identification using an enzyme-labeled second antibody. In addition, we will discuss two other applications of protein blotting that provide rapid but less precise results. These are analysis of tissue extracts using dot blotting, and “squash” blots of whole plants or plant organs.