Since the purity of the reagents is critical to the success of the experiment, the following steps are critical if the antibodies used are not blotting-grade: Addition of a suitable enzyme substrate results in production of a colored precipitate or fluorescent or chemiluminescent product through dephosphorylation (by AP) or oxidation (by HRP). If the secondary antibody is biotinylated, biotin-avidin-AP or -HRP complexes can be formed. The antibody can be radiolabeled or linked to a fluorescent compound or to gold particles, but most commonly, the antibody is conjugated to an enzyme, such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). Secondary antibodies can be labeled and detected in a variety of ways. For immunodetection, only blotting-grade species-specific secondary antibodies are used. Secondary antibodies bind to multiple sites on primary antibodies to increase detection sensitivity. Secondary antibodies are specific for the isotype (class) and the species of the primary antibody (for example, a goat anti-rabbit secondary antibody is an antibody generated in goat for detection of a primary antibody generated in a rabbit). The Mini-PROTEAN ® II multiscreen apparatus and mini incubation trays are useful tools for determining antibody titer. 1:1,000–1:100,000 dilution may be used when ascites fluid is the source of antibodyįor each new lot of primary antibody, determine the appropriate concentration or dilution (titer) empirically.1:500–1:10,000 dilution of chromatographically purified monospecific antibodies.1:100–1:1,000 dilution of the primary antibody in buffer when serum or tissue culture supernatants are the source of the primary antibody.For custom antibodies, or those where a dilution range is not suggested, good starting points are: Instructions for antibodies obtained from a manufacturer typically suggest a starting dilution range. ![]() ![]() The optimum antibody concentration is the greatest dilution of antibody that still yields a strong positive signal without background or nonspecific reactions. For successful incubations with primary antibodies, the entire blot must be covered with antibody-containing solution. The primary antibody recognizes and binds to the target antigen on the membrane.
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