Goat Anti-Rabbit IgG (H+L) Secondary Antibody, Cy3 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, and then modified with antibody fragmentation, label conjugation, etc., to generate highly specific detection reagents. Secondary antibodies can be conjugated to a large number of labels, including enzymes, biotin, fluorescent dyes/proteins, or gold particles. Here, the antibody provides the specificity to locate the protein of interest by recognizing a primary antibody that targets a particular antigen, and the label generates a detectable signal in the area of the formed immune complexes. The label of choice depends upon the experimental application.

Fluorescent reporters widely used in biological research are of two types: organic compounds with a low molecular weight (0.2-1 kDa) typically containing numerous aromatic groups, or plane or cyclic moieties with π bonds (e.g.FITC, TRITC, Alexa Fluor Dyes, DyLight Fluors), and biological fluorophores (e.g.green fluorescent protein (GFP), R-Phycoerythrin). Cy3 is a cyanine dye with 3 carbon atoms/methine groups between two indolenine groups, NHS-ester, sulfonated at both indoles. It fluoresces greenish yellow with excitation max at ~550 nm and emission at ~570 nm. Cy3 can be detected by various fluorometers, imagers, and microscopes with standard filters for Tetramethylrhodamine (TRITC). Due to inherently high extinction coefficient, this dye is also easily detected by naked eye on gels, and in solution. Cy3 can be excited to about 50% of maximum with an argon laser (514 nm or 528 nm lines), or to about 75% of maximum with a helium/neon laser (543 nm line) or mercury lamp (546 nm line). For double labeling Cy3 has been typically used with Cy5, with fluorescein (using narrow band-pass emission filter to minimize Cy3 fluorescence in the FITC filter set), and also with with Alexa Fluor 647 for multiple labeling when using a confocal microscope. Analog of Cy3 with greater photostability and higher fluorescence intensity suited for various biological applications such as imaging and flow cytometry, is Alexa Fluor 555 dye.

Validation Images & Assay Conditions

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GFAP was detected in a paraffin-embedded section of rat brain tissues using rabbit anti-GFAP Antigen Affinity purified polyclonal antibody (Catalog # PB9082). Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) was used to detect the primary antibody.

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PCNA was detected in immunocytochemical section of A549 cells using rabbit anti-PCNA Antigen Affinity purified polyclonal antibody (Catalog # A00125). Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) was used to detect the primary antibody.