Fluorescent microscope
Fluorescein isothiocyanate, commonly referred to as FITC -- pronounced "fit-c," to rhyme with "ditsy" -- is a fluorescent marker that is chemically bound, or conjugated, to other molecules such as proteins and antibodies. An antibody conjugated with FITC might also be referred to as a "FITC-labeled antibody." FITC is excited by light at a wavelength of 488 nm and gives off fluorescent energy detected at 530 nm. A scientist can see it with a fluorescent microscope or use equipment that detects the fluorescent emissions and records them.
Methods
FITC conjugation follows a basic protocol. For example, FITC and an antibody would be mixed together at a certain ratio in a test tube and rotated at room temperature for an hour. To remove the unbound FITC from the solution, it is poured onto an affinity chromatography column -- a glass tube filled with a bead-like gel that will bind the unconjugated FITC. Meanwhile, the FITC-labeled antibody will flow through the column and is collected for use.
Considerations
Before starting a conjugation, the researcher should consider several points. First, FITC is highly unstable and should not be exposed to light -- it should be kept in foil-covered tubes at all times. Second, the conjugation time and temperature may have to be altered for a temperature-sensitive protein. Finally, different proteins and antibodies can have different FITC-binding capacities, so finding the correct ratio of antibody to FITC is critical; too little FITC makes an antibody undetectable and too much can alter its specificity. For this reason, preliminary small-scale conjugations at different ratios of FITC to antibody should be set up in parallel. Once the optimal ratio is established, the large-scale conjugation can be performed.
Uses
Doctors and scientists use FITC-labeled antibodies, hormones and growth factors in a wide variety of assays and applications. For instance, a stem cell found in the blood has a molecule on its surface called CD34. A lab technician can mix a patient's blood with a FITC-labeled antibody specific for CD34, and run the cells through a flow cytometer that uses a laser beam to make the FITC fluoresce. Next, the cytometer separates the fluorescent CD34 cells from the rest of the blood cells, and doctors can use these isolated stem cells for therapeutic treatments.
Tags: FITC-labeled antibody, antibody FITC, CD34 cells, FITC antibody, proteins antibodies
