secondary antibody review

secondary antibody review

See ExactAntigen.com for reviews on primary antibodies. For example, actin antibody, tubulin antibody, integrin antibody, stem cell markers, phosphotyrosine antibody, and hemagglutinin antibody.

overview

In order to help Secondary-Antibody.com visitors identify the most suited secondary antibodies, information about secondary antibodies from 99 recent publications in Science and Journal of Biological Chemistry were collected and catalogued. The publications were randomly selected. Their authors came from more than 80 universities or research institutes in 16 countries. Data include host species, reactive species, Ig class, Ig subclass, conjugation, experimental methods, antibody dilution, and suppliers. Quite often, the relevant information are not available in these articles. In such cases, emails were sent to the corresponding authors and their clarifications are incorporated.

summary

host and reactive species: Primary antibodies tend to be raised in goats, mice, rabbits, rats, and sheep, with mice and rabbits are the most common ones. The reactive species of secondary antibodies correlate well with the host species of primary antibodies, with rabbits and mice as dominant types.

Production of secondary antibodies requires species different from those of primary antibodies. Goats, donkeys, rabbits, sheep, and mice (for monoclonal secondary antibodies) are the hosts of choice for secondary antibodies.

Ig class, subclass, and secondary antibody form: Anti-IgG secondary antibodies are most widely used. This is not surprising since most monoclonal and polyclonal primary antibodies are IgG class immunoglobulins. In addition, anti-IgG secondary antibodies can recognize various IgG subtypes. If the class and/or subclass of a primary antibody is unknown, an anti-IgG secondary antibody may be a good default choice.

Secondary antibodies specific to IgG subclasses (e.g. anti-IgG1, anti-IgG2, anti-IgG2a, anti-IgG2b) appear to be mainly used in double labeling experiments, including immunocytochemistry, immunohistochemistry, and flow cytometry.

Secondary antibodies tend to be against whole IgG molecule. Those agasint F(ab')2 fragments or Fab fragment are used, for instances, when binding of secondary antibodies to Fc receptors on cell surfaces should be avoided, or there is endogenous immunoglobulin.

secondary antibody applications: Secondary antibodies are used in all types of immunoassays, most often in Western blot, immunohistochemistry, and immunocytochemistry, and occasionally in immunoprecipitation. Basic research, clinical analysis, and disease diagnosis also use secondary antiobodies in ELISA and flow cytometry assays.

conjugate: The choice of conjugates of secondary antibodies depends on specific application. For Western blot, the conjugate is likely to be horseradish peroxidase (HRP), alkaline phosphatase (AP), and fluorescent dyes like IRDye (e.g. IRDye 680, IRDye 800) and Alexa Fluor dye (e.g. Alexa 680 ) series of conjugates. HRP conjugated secondary antibodies from Amersham Biosciences, Bio-Rad, Cell Signaling, GE Healthcare Bio-Science, Jackson ImmunoResearch Laboratories, LI-COR Biotechnology, Pierce Biotechnology, Santa Cruz Biotechnology, Sigma, and Invitrogen(Zymed Laboratories) are often used in Western blot, and AP conjugated secondary antibodies are from Jackson ImmunoResearch Laboratories, Sigma, Bio-Rad, and DAKO.

For immunofluorescence (immunocytochemistry and immunohistochemistry), Alexa Fluor dye series of conjugates (e.g. Alexa Fluor 488, 546, 568, 594, 596, 647, 633), Cyanine dye series of conjugates (e.g. Cy2, Cy3, and Cy5), FITC, and rhodamine red are likely used, with Invitrogen, Jackson Immunoresearch Laboratories, and Sigma as the main suppliers.

Secondary antibodies conjugated with FITC, PE, or Alexa Fluor 647 are used in flow cytometry; those conjugated with HRP, AP, and biotin are used in ELISA assays; and unconjugated secondary antibodies are used for protein isolation in immunoprecipitation.

secondary antibody suppliers: Many high quality reagent providers supply secondary antibodies to both the research and diagnostics community. The relatively small number of publications in this survey precludes an extensive sampling of suppliers. Invitrogen (including Molecular Probes, Biosource International, and Zymed Laboratories), Jackson ImmunoResearch Laboratories, Sigma, GE Healthcare Bio-Science (including Amersham Biosciences), Bio-Rad, Santa Cruz Biotechnology are the most cited suppliers.

Secondary Antibody