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FITC Conjugated Antibodies & Reagents

First described by Albert H. Coons in 1942, fluorescein isothiocyanate (FITC) is one of the most widely used dyes for fluorescence-based detection. Each FITC molecule consists of a fluorescein core with an isothiocyanate functional group, which allows for covalently attaching fluorescein to antibodies and other amine-containing biomolecules.

Advantages of FITC include its bright fluorescence, high water solubility, and low non-specific binding with biological tissues. FITC is also relatively inexpensive and has a vast number of literature citations as a result of its long-established history.

FITC-conjugated antibodies have utility for applications including flow cytometry, immunocytochemistry (ICC), and immunohistochemistry (IHC), where they are often combined with other fluorophore-labeled antibodies to enable multicolor detection. When performing these types of experiments, selecting compatible fluorophores is important to avoid spectral overlap that could lead to data being misinterpreted.

We offer an extensive selection of FITC-conjugated primary and secondary antibodies, in addition to FITC-conjugated isotype controls and streptavidin-FITC conjugates.

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Frequently Asked Questions

FITC has an excitation maximum of 492 nm and an emission maximum of 518 nm. It can be excited with the 488 nm laser line and is commonly detected with a 530/30 filter set.

One of the main reasons that FITC is popular for fluorescent detection is that its excitation maximum closely matches the 488 nm laser line, found in most instruments. FITC also has a reasonably large extinction coefficient (7.3 x 104 M-1 cm-1) and a high quantum yield (0.92), resulting in a relatively strong fluorescence signal. Another important advantage of FITC is its versatility; the isothiocyanate group reacts with primary amines, which are abundant on antibodies and other biomolecules. In addition, FITC has high water solubility, exhibits low non-specific binding with biological tissues, and produces conjugates that are relatively stable, as well as benefits from being both inexpensive and widely literature-cited.

A limitation of FITC is that it exhibits a relatively high rate of photobleaching. However, when performing ICC and IHC staining, this can be mitigated by including an anti-fading agent in the mounting medium. Additionally, the fluorescent signal emitted by FITC is sensitive to changes in pH and solvent polarity, although careful optimization of the staining conditions can often address this issue.

Like other dyes with similar emission spectra to FITC, the fluorescent signal can be masked by autofluorescence during microscopy-based applications such as ICC and IHC. Strategies for reducing autofluorescence include titrating FITC-conjugated antibodies to increase the signal-to-noise ratio, switching from an aldehyde-based fixative to methanol fixation, and introducing an autofluorescence reducing treatment (e.g., ultraviolet light, Sudan Black B, or sodium borohydride) into the experimental workflow.

When performing multicolor detection, it is important to select fluorophores with distinct emission spectra. This is to avoid spectral overlap that could lead to results being misinterpreted. Because FITC emits green fluorescence, fluorophores that emit other colors than green are generally preferred. For example, PE is often used in combination with FITC, provided it is compatible with the instrument's lasers and detectors.

Because FITC exhibits a relatively high rate of photobleaching, it is not typically used for long-duration imaging experiments. Alexa Fluor® 488, which has an excitation maximum of 490 nm and an emission maximum of 525 nm, represents a more viable alternative for these types of studies. As well as being less prone to photobleaching than FITC, Alexa Fluor® 488 can better resist pH changes.

FITC-conjugated antibodies and reagents should be stored at 2-8°C, avoiding exposure to light. They can also be aliquoted into smaller volumes, to avoid freeze/thaw cycles, and stored at -20°C.

Related Product Categories

Conjugated Antibodies

For the direct detection of your target, explore our offering of over 25 enzyme and fluorochrome conjugated primary antibodies.

Flow Cytometry Reagents

Maximize fluorescence signal intensity and minimize non-specific binding in your flow cytometry assays.

Isotype Controls

Move quickly and keep your assay development high quality and on track with our wide selection of isotype controls.

Secondary Antibodies & Reagents

With a wide range of ready-to-ship secondary antibodies that are second to none, SouthernBiotech will keep your assays running and projects on schedule.