Western blotting is a technique that is a cornerstone in protein research. It is used extensively to detect and quantify proteins in complex mixtures. Western blotting actually starts with gel electrophoresis
of the sample. Once the proteins are separated in the gel, they are transferred or blotted to a membrane via electrophoresis (Figure 1). The gel and membrane are sandwiched between blotting papers soaked in buffer, and the set is compressed between two parallel electrodes in a cassette. Current is passed at right angles to the gel, causing the proteins in the gel to migrate towards the membrane.
Once the proteins have been transferred to the membrane (referred to as blot
), they can be detected by several methods, such as chemiluminescence, fluorescence, chromogenic, and autoradiography. The detection step usually involves probing the blot using an antibody to detect a specific protein. The blot is first incubated in a blocking solution, for example 5% (w/v) non-fat dry milk or 10% (w/v) bovine serum albumin. This step will block all remaining binding sites on the membrane. The blot is next incubated in a dilution of antiserum (the primary antibody
), that is directed against the protein of interest. After several washing steps, the blot is incubated with a labeled secondary antibody, which is directed against the species that provided the primary antibody. The label on the secondary antibody provides a way to visualize the interaction of the secondary antibody with the primary antibody on the blot. The label could be a fluorescent molecule, isotopic labels, and more commonly, enzymes. When an enzyme-labeled secondary antibody is used, the blot is incubated in a substrate which gets converted to a product that can be detected (e.g.
light, colored precipitate).
Figure 2 summarizes the workflow for Western blotting.
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