b'Secondary antibody incubation as well as any other molecules sharing the same conserved A secondary antibody aids in the detection of a targetdomains (e.g., IgM shares the same kappa light chains protein by binding to a primary antibody that has beenas IgG). In contrast, immunizing a goat with only mouse directly bound to the target protein. The vast majority ofIgG1 antibodies will only generate antibodies specific for primary antibodies are produced in just a few host animalmouse IgG1 antibodies and molecules sharing the same species and most are of the IgG class, making it relativelyconserved domains. easy to choose ready-to-use, labeled secondary antibodies for most applications and detection systems. SecondaryBecause of the high degree of conservation in the antibodies can be polyclonal, monoclonal, or recombinant.structure of many immunoglobulin domains, class-specific They are available in different buffer formulations andsecondary antibodies must be affinity-purified and forms, for example lyophilized or in solution. The secondarycross-adsorbed to achieve minimal cross-reaction with antibody incubation step in western blotting is usuallyother immunoglobulins. Using the example described performed for 1 hour at room temperature in blockingabove, immobilized mouse IgG1 antibodies would be used buffer. Longer incubations can be used, if needed.to affinity-purify all goat antibodies that bind to mouse IgG1. These antimouse IgG1 antibodies would then be further Secondary antibodies for western blotting are typicallypurified by passage through one or more chromatography diluted to working concentrations ranging from 1:500columns containing mouse IgG2a, IgG2b, IgG3, IgM, and for fluorescent western blotting to 1:500,000 forother isotypes to remove any antibodies that cross-react chemiluminescent detection of abundant targets whenwith non-IgG1 isotypes. Additionally, secondary using a substrate designed for high sensitivity. Theantibodies can be further purified by passage through optimal dilutions vary significantly between fluorescentcolumns containing the immobilized serum proteins from and chemiluminescent detection. Optimal dilutionspecies other than those used to immunize the host. for chemiluminescent detection depends on severalThis method of cross-adsorption (typically referred to experimental factors, such as protein target abundance,as cross-adsorbed in product names) is an additional choice of primary antibody, and, most importantly, choicepurification step recommended for applications where of the chemiluminescent substrate. Dilutions are typicallyprimary antibodies from multiple species will be used and higher for enzyme-conjugated secondary antibodies duewhen immunoglobulins or other serum proteins may be to enzymatic amplification of the chemiluminescent signal.present in the samples being probed (Table 14).We offer a wide variety of labeled secondary antibodies for use in western blotting. The labels include horseradishTable 14. Commonly used abbreviations for peroxidase (HRP) and alkaline phosphatase (AP) fortarget species.chemiluminescent and chromogenic detection, poly-HRPTargetTarget and biotin conjugates for signal amplification, andspecies Abbreviation species AbbreviationInvitrogen Alexa Fluor and Alexa Fluor Plus secondaryBovine Bv HumanHuantibodies for fluorescent western blotting.CanineCa HorseEqChickenCk MonkeyNhpSpecificity of secondary antibodies DonkeyDo MouseMsSecondary antibodies are generated by immunizing aFelineFe RabbitRbhost animal with an antibody from a different species.GoatGt RatRtFor example, anti-mouse antibodies can be raised byGuinea pigGP SheepOvinjecting specific purified mouse antibody into an animalHamsterHm PigPoother than a mouse. Goat, donkey, sheep, chicken, and rabbit are the most commonly used host species for raising secondary antibodies, though others are available.Enzyme labels for detection: alkaline phosphatase (AP) The most common types of secondary antibodiesand horseradish peroxidase (HRP) are those generated against a pooled population ofEnzymatic labels are most commonly used as secondary immunoglobulins from a target species. For example,antibody tags for detection in western blotting applications. immunizing a goat with purified mouse IgG will generateEnzymes provide detectable signal via their activity; goat antimouse IgG antibodies that will bind to all classes,reaction with a specific substrate chemical yields a heavy and light chains (H+L), and fragments of mouse IgG,colored or chemiluminescent product. AP and HRP 36'