Goat Anti-Human IgA Secondary Antibody, FITC Conjugated

Product Overview

Product Name	Goat Anti-Human IgA Secondary Antibody, FITC Conjugate
Synonyms	FITC-conjugated Goat Anti-Human IgA; Goat Anti-Human IgA-FITC Secondary Antibody; Fluorescein-labeled Goat Anti-Human IgA Secondary Antibody
Description	Goat Anti-Human IgA Secondary Antibody, FITC Conjugate, for detection, localization and quantification of target proteins in a sample via indirect immunofluorescence in IHC-P, IHC-F, ICC, or FCM
Reagent Type	Fluorophore-conjugated secondary antibody
Label	FITC
Host	Goat
Target Species	Human
Antibody Class	IgG
Clonality	Polyclonal
Immunogen	Whole molecule human IgA
Purification	Immunoaffinity chromatography
Specificity	Human IgA specific; No cross-reactivity with human IgG/IgM
Form Supplied	Liquid, concentrated buffered stock solution
Formulation	0.5 mg FITC-conjugated secondary antibody 0.01 M PBS (PH 7.4) 5 mg/mL BSA 50% glycerol
Pack Sizes	0.25 ml, 0.5 ml
Concentration	1 mg/ml
Application	IF, Flow Cytometry *Our Boster Guarantee covers the use of this product in the above marked tested applications.
Storage	At -20˚C for one year from date of receipt. Avoid repeated freezing and thawing. Protect from light.
Precautions	FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR CLINICAL USE

Assay Information

Sample Type	Human primary-antibody-probed Single cell suspension, Formalin-fixed paraffin-embedded (FFPE) tissue sections, Thawed frozen samples (IHC-F)
Assay Type	Immunoanalytical
Assay Purpose	Protein detection/quantification
Technique	Immunofluorescence
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
Additional Materials Required	Primary antibody against target antigen raised in human; Diluent Buffer (PBS or TBS); Application-specific reagents and appliances;

Main Advantages

Specific	High signal-to-noise ratio
High Signal Amplification	Multiple secondary antibodies can bind to a single primary antibody; Multiple FITC 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; Fluorescence can be observed directly
Quantifieable	Allows quantification of detected signal
Easy to Use	Supplied in a workable liquid format
Multiplex Compatibility	Colocalization studies possible, even in close proximity: use primary antibodies from different host species for simultaneous detection by fluorophore-conjugated secondary antibodies; use multiple differently colored fluorophores in the same experiment for target differentiation
Dynamic range	Good linearity within detection limits

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