b'Transfer of animal in which the primary antibody was raised (the host Following electrophoresis, the proteins must be transferredspecies) or any tag on that antibody (e.g., biotin). from the polyacrylamide gel to a membrane, typically usingFollowing the blocking and antibody incubations steps, electroelution, or electrophoretic transfer, because of its speedextensive washing is necessary to remove unbound reagents and transfer efficiency. This method uses the electrophoreticand reduce background, thereby increasing the signal-to-noise mobility of proteins to transfer them from the gel to theratio. Insufficient washing will result in high background, while membrane. Electrophoretic transfer of proteins involves placingexcessive washing may result in decreased sensitivity caused by the gel in direct contact with a piece of nitrocellulose or otherelution of the antibody and/or antigen from the blot. As with other suitable protein-binding support and sandwiching this betweensteps in western blotting, a variety of buffers may be used.two electrodes submerged in a conducting solution. When an electric field is applied, the proteins move out of the gel andDetection steponto the surface of the membrane, where the proteins becomeWhile there are many different tags that can be conjugated to tightly attached. The result is a membrane with a copy of thea secondary or primary antibody, the detection method used protein pattern that was originally in the gel. Efficiency can varywill limit the choice of what can be used in a western blotting dramatically among proteins; it also depends on factors such asassay. Alkaline phosphatase (AP) and horseradish peroxidase the composition of the gel, degree of contact of the gel with the(HRP) are the two enzymes used most extensively as labels membrane, the position of the electrodes, the transfer time, sizefor protein detection. An array of chromogenic, fluorogenic, and composition of proteins, field strength, and the presence ofand chemiluminescent substrates are available for use with detergents and alcohols in the buffer. either enzyme. HRP-conjugated antibodies, which generally use chemiluminescent substrates, are considered superior to Blocking and washing AP-conjugated antibodies with respect to the specific activities The membranes used in western blotting have a high affinity forof both the enzyme and antibody, due to the smaller size of proteins. Therefore, after the transfer of proteins from the gel,HRP and compatibility with conjugation reactions. In addition, it is important to block the remaining surface of the membranethe high activity rate, good stability, low cost, and wide to prevent nonspecific binding of detection antibodies duringavailability of substrates make HRP the enzyme of choice for subsequent steps. The blocking buffer should improve themost applications. In well-optimized assays, chemiluminescent sensitivity of the assay by reducing background interference andreactions can produce stable light output for 124 hours, improving the signal-to-noise ratio. depending on the substrate, allowing consistent and sensitive detection that may be documented with X-ray film or digital Use of antibodies imaging equipment.Western blotting is typically performed by probing the blockedThe use of fluorophore-conjugated antibodies requires fewer membrane with a primary antibody that recognizes a specificsteps because there is no substrate development step in the protein or epitope. The choice of primary antibody for a westernassay. Recent advances in digital imaging and development of blot will depend on the antigen to be detected and the availabilitynew fluorophores have increased the sensitivity and popularity of antibodies for that antigen that have been verified for westernof using fluorescent probes for western blotting and other blotting. In general, a primary antibody that recognizes the targetimmunoassays. This method has the added advantage of protein is not directly detectable. Therefore, tagged secondarymultiplex compatibility (using more than one fluorophore in the antibodies or other detection reagents are used as the meanssame experiment).of ultimately detecting the target protein (indirect detection). A wide variety of labeled secondary detection reagents can be used; the choice of which depends upon either the species 58 Western blotting thermofisher.com/proteinbiology'