Fluorescein isothiocyanate (FITC) is a widely used due to its high quantum efficiency and stability when conjugated. FITC is yellow-orange in color with an absorption maximum at 495 nm. Upon excitation it emits a yellow-green color with an emission maximum at 525 nm. Conjugation occurs through primarily-amine groups of lysine residues, forming a stable thiourea bond. Conjugation of fluorescein isothiocyanate (FITC) to antibody is an extremely valuable technique for identifying surface molecules using either fluorescence microscopy or flow cytometry. The following is the procedure that antibody molecules are coupled with fluorescein derivatives.
1. Change the antibody solution with 0.2 M Sodium carbonate-bicarbonate buffer (pH 9.0) with G-25 column.
2. Determine antibody concentration based upon A280.
3. Prepare the required dilution of FITC in 0.2 M Carbonate-bicarbonate buffer (FITC labeling buffer).
4. Add FITC labeling buffer to antibody solution and standing for overnight at 4°C.
5. Cover the reaction vial with aluminum foil to protect from light.
6. Separate conjugate from free FITC on G-25 column, and collect fractions.
7. Determine F/P ratio of conjugate spectrophotometrically.
F/P = Moles of FITC / Moles of protein
An F/P of 5 to 6:1 is usually optimal for flow cytometry.
8. Change antibody solution with Phosphate buffered saline (PBS) buffer with G-25 column.
9. Collect the labeled solution. Store at 4°C and protect from light.