Rabbit Anti-Goat IgG (H+L) Secondary Antibody, FITC 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.

Immunofluorescence is a technique used for light microscopy with a fluorescence microscope which utilizes fluorescent dyes as reporters. It is being employed in a variety of applications such as cellular imaging and flow cytometry and is commonly used to visualize the distribution of target molecules through a sample, to detect protein location and activation, to identify protein complex formation and conformational changes, and to monitor biological processes in vivo. Fluorescent dyes (also known as fluorochromes, fluorophores, or simply fluors) are molecules that can absorb light of a specific energy and wavelength, thereby undergoing excitation, and then re-emit it at a lower energy and longer wavelength upon returning to the ground state.

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). FITC (Fluorescein isothiocyanate) is derivative of fluorescein where a hydrogen atom on the bottom ring of the structure is replaced by isothiocyanate functional group (-N=C=S), making it more reactive to amine and sulfhydryl groups on proteins. The excitation and emission wavelengths of FITC are 495 nm and 525 nm respectively. Like most fluorochromes, it is prone to photobleaching, i.e. losing fluorescing properties due to molecule structure degradation. Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of the fluorophore, or by employing more robust fluorophores that are less prone to bleaching (e.g., Alexa Fluors, Seta Fluors, or DyLight Fluors). Analogs of FITC with greater photostability and higher fluorescence intensity tailored in various biological applications are Alexa 488 and DyLight 488.

Validation Images & Assay Conditions

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Boster Kit Box