Goat Anti-Mouse IgG (H+L) Secondary Antibody, TRITC 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). TRITC (tetramethylrhodamine isothiocyanate )is a bright orange-fluorescent dye. It is a derivative of rhodamine (a member of the fluorone dyes family) 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 bond to antibodies is based on this reactive group. The excitation and emission wavelengths of TRITC are 550 nm and 573 nm respectively. The optimal degree of conjugation for least changes in the antibody affinity and maximal specific staining (fluorescence) is at a molecular ratio TRITC/Ab of approximately 2. Like most fluorochromes, TRITC is prone to photobleaching, i.e. losing fluorescing properties due to molecule structure degradation. TRITC is inherently a quite stable dye, and thus exhibits less photobleaching than fluorescein, which is commonly used as a photobleaching standard.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 TRITC with greater photostability and higher fluorescence intensity tailored in various biological applications are Alexa 555 and DyLight 550.

Validation Images & Assay Conditions

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Figure 1. IHC analysis of rat heart using anti-Alpha-Smooth Muscle Actin antibody (MA1106).
Alpha-Smooth Muscle Actin was detected in paraffin-embedded section of rat heart tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 10μg/ml mouse anti-Alpha-Smooth Muscle Actin Antibody (MA1106) overnight at 4°C. TRITC Conjugated goat anti-mouse IgG (BA1089) was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1021) with DAB as the chromogen.