A fluorescein isothiocyanate-conjugated (FITC) antibody is a fluorescent molecular marker highly specific for either a particular protein or a primary antibody. They are used to identify proteins in immunocytochemistry, enzyme and protein activity assays, flow cytometry, and Western blotting. Most FITC antibodies can be purchased through specialized molecular biology companies or made by individual research labs.
FITC is a fluorochrome often conjugated or linked to an antibody in order to make a specific, fluorescent marker. When exposed to electromagnetic energy at a specific wavelength, fluorochromes are chemicals that will absorb the energy and emit a photon at a specific wavelength. FITC absorbs energy at 495 nm and emits a photon at 521 nm. Each fluorochrome has a different absorption and emission range, allowing for systematic detection of many fluorchrome-linked antibodies in one piece of tissue or sample. This is similar to labeling different parts of a cell with different colors and then using specialized glasses to look at just one color at a time.
Antibodies can be linked, or conjugated, to the FITC molecule to produce a FITC antibody. This FITC antibody can be used to directly or indirectly label a protein. When a FITC antibody directly labels a protein, it acts as the primary antibody, or an antibody in direct contact with the antigen. Indirect use of a FITC antibody occurs when it is used as a secondary antibody, binding specifically to a primary antibody. When a FITC conjugated antibody is used directly, the labeling procedure takes less time, but the labeling is not as strong. Indirect labeling takes an extra step, but the signal or label is amplified because several FITC-tagged secondary antibodies can attach to a single primary antibody.
There are many uses for the FITC antibody in cell and molecular biology. This type of antibody can be used alone or in combination with other monoclonal or polyclonal antibodies to label specific proteins within cells in a technique known as immunocytochemistry. FITC antibodies can also be used in such techniques as enzyme and protein activity assays, flow cytometry, and Western blotting.
FITC conjugated primary and secondary antibodies are widely available through many molecular biology companies. It is also possible for labs to prepare their own primary antibodies and then use a kit to conjugate FITC to them. Some companies will take lab-prepared primary antibodies and conjugate them to the FITC fluorochrome for a lab. FITC-conjugated secondary antibodies are not typically made by individual research labs, and are usually purchased from companies specializing in the production of secondary antibodies.
