Protein Electrophoresis and Blotting
Proteins in a mixture (e.g. cell culture lysate, tissue homogenate, etc.) are separated by molecular weight, or isoelectric point, using polyacrylamide gel electrophoresis (PAGE). The proteins are transferred (blotted) from the gel to a membrane (nitrocellulose or PVDF) for easier handling and manipulation.
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Labeling
Following the blotting step, the target protein is labeled using antibodies. The primary antibody, which is specific for the target protein, can be labeled or unlabeled. To maximize sensitivity and signal-to-noise ratio, most Western blotting procedures use an unlabeled primary antibody and a conjugated or labeled secondary antibody (the secondary antibody is specific for the primary antibody). The secondary antibody can be radiolabeled, labeled with a dye or other molecule, or conjugated with an enzyme. Typical enzymes used are horse radish peroxidase (HRP) and alkaline phosphatase (AP), both of which use a detection reagent to generate a signal that can be quantitated. The signal can be the production of an insoluble dye (chromogenic) or the generation of light (chemiluminescent or chemifluorescent).
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Detection and Image Analysis
Target proteins are detected using the appropriate detection reagents to generate a signal that can be quantitated. For chromogenic methods, the signal is captured directly on the membrane. For radiolabeling, chemiluminescent, and chemifluorescent methods, the signal is captured using film or an imaging system. The acquired image is quantitated using image analysis software.
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