|Product Name||Goat Anti-Rabbit IgG（H+L） Secondary Antibody, TRITC Conjugate|
|Synonyms||TRITC-conjugated Goat Anti-Rabbit IgG; Goat Anti-Rabbit IgG-TRITC Secondary Antibody; Rhodamine-labeled Goat Anti-Rabbit IgG Secondary Antibody|
|Description||Goat Anti-Rabbit IgG Secondary Antibody, TRITC Conjugate, for detection, localization and quantification of target proteins in a sample via indirect immunofluorescence in IHC-P, IHC-F or ICC.|
|Reagent Type||Fluorophore-conjugated secondary antibody|
|Immunogen||Whole molecule rabbit IgG|
|Specificity||Rabbit IgG specific|
|Form Supplied||Liquid: concentrated buffered stock solution|
|Formulation||0.5 mg TRITC-conjugated secondary antibody |
0.01 M PBS (PH 7.4)
|Application||Immunohistochemistry (IHC-P and IHC-F), Immunocytochemistry (ICC) |
|Storage||4C for 1 year|
|Precautions||FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR CLINICAL USE|
|Sample Type||Rabbit primary-antibody-probed formalin-fixed paraffin-embedded (FFPE) tissue sections on slides (IHC-P), or thawed frozen samples (IHC-F)|
|Assay Purpose||Protein detection/quantification|
|Equipment Needed||Excitation light source, filter set and detector, fluorescence microscope (can be combined with confocal microscope), fluorescence plate-reader, flow cytometer, or cell sorter|
|Specific||High signal-to-noise ratio|
|High Signal Amplification||Multiple secondary antibodies can bind to a single primary antibody, multiple TRITC molecules bind to a single secondary antibody|
|Fast||Fewer processing steps - no need to add a substrate; Less optimization required compared to enzymatic detection; Generates strong signals in a relatively short time span; signal can be observed directly|
|Quantifieable||The digital nature of the gold signal + high precision in allocating gold labels to defined structures makes it easy to count and quantify|
|Flexible||No need to label each antibody against each target protein with a fluorescent dye, the small size of TRITC causes no steric interference with proper biological function of target proteins or antibodies|
|Multiplex Compatible||Compatible with colocalization studies (multiple antigens concurrent detection) even in close proximity using primary antibodies from different host species for simultaneous detection by fluorophore-conjugated secondary antibodies, or using multiple differently colored fluorophores (FITC and TRITC) in the same experiment for target differentiation.|
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.
Cpn10 was detected in paraffin-embedded sections of human mammary cancer tissues using rabbit anti- Cpn10 Antigen Affinity purified polyclonal antibody at 1 μg/mL. TRITC Conjugated Goat Anti-rabbit IgG secondary antibody (Catalog # BA1090) was used to detect the primary antibody at 30 μg/mL.
MCM2 was detected in paraffin-embedded sections of human intestinal cancer tissues using rabbit anti- MCM2 Antigen Affinity purified polyclonal antibody at 1 μg/mL. TRITC Conjugated Goat Anti-rabbit IgG secondary antibody (Catalog # BA1090) was used to detect the primary antibody at 30μg/mL.