Rabbit Anti-Rat IgG (H+L) Secondary Antibody, Biotin Conjugated

Product Overview

Assay Information

Main Advantages

Background

Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species. The host antiserum is then purified through immunoaffinity chromatography to remove all host serum proteins, except the specific antibody of interest. Purified secondary antibodies are further solid phase adsorbed with other species serum proteins to minimize cross-reactivity in tissue or cell preparations, and are then modified with antibody fragmentation, label conjugation, etc., to generate highly specific reagents. Secondary antibodies can be conjugated to a large number of labels, including enzymes, biotin, and fluorescent dyes/proteins. Here, the antibody provides the specificity to locate the protein of interest, and the label generates a detectable signal. The label of choice depends upon the experimental application.

Biotinylated antibodies are widely used in systems where signal amplification is desired. Often 15-20 biotin moieties are coupled to a single IgG secondary antibody. Biotin binds avidin, streptavidin, or neutravidin with a high degree of affinity and specificity. In immunoassays avidin/streptavidin-biotin binding is used as a bridge between antibodies and reporters like enzymes (HRP, AP), fluorophores, chromophores, etc. Both avidin and streptavidin are tetrameric proteins capable of binding 4 biotin groups to each molecule of avidin or streptavidin, thus amplifying the signal intensity and detection sensitivity by increasing the concentration of reporters at the antigenic site. Two main biotin-binding detection systems have been widely utilized: Avidin-Biotin Complex (ABC) and Labeled Streptavidin Biotin (LSAB) methods. In the ABC method free avidin (or streptavidin) is used as a bridge/link between the biotinylated antibody and а biotinylated reporter molecule, resulting in three reporter molecules coupled to the biotinylated antibody. The LSAB method employs a reporter-labeled streptavidin (avidin or neutravidin can alternatively be used) to detect the bound biotinylated-secondary antibody on the tissue section, blotting membrane or ELISA plate, improving the sensitivity of detection by 8-fold. The LSAB method is used when the avidin-biotin-enzyme complex in the ABC method becomes too large to penetrate the tissue.

Validation Images & Assay Conditions

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Boster Kit Box