FITC (fluorescein isothiocyanate) is a fluorochrome dye widely used as an antibody or other probe marker. FITC absorbs ultraviolet or blue light, exciting molecules which then emit a visible yellow-green light. When the excitation light is removed, the emission light ceases. FITC has high fluorescence with excitation and emission peak wavelengths at about 495nm and 525nm. FITC conjugation to proteins is relatively simple, and usually does not alter the biological activity of the labeled protein. Invitrogen anti-FITC antibodies reliably detect FITC and can be useful when double-labeling if one of the existing antibodies is only available as a FITC conjugate. Anti-FITC antibodies can also be useful in qualifying successful FITC conjugation. Our FITC antibodies come with the option of being monoclonal, polyclonal, or recombinant and available conjugated or unconjugated.
Invitrogen anti-FITC antibodies are designed to dependably detect FITC in various applications.
Flow cytometry using anti-FITC antibody. Fixed and permeabilized A549 cells were labeled with mouse anti-tubulin antibody (1 µg/mL), followed by detection using a FITC-conjugated goat anti-mouse IgG secondary antibody (right peak, green). To confirm specificity, the cells were labeled with a mouse isotype control and stained using FITC goat anti-mouse IgG (middle peak, pink). Quenching of fluorescence upon pre-incubation of 1:100 of FITC Recombinant Polyclonal Antibody (Cat. No. 710083) is shown as middle peak, red. Unstained control cells are as shown (left peak, purple). The representative 10,000 cells were obtained and analyzed for each sample using Attune NxT Flow Cytometer.
Western blot analysis of fluorescein isothiocyanate (FITC) antibody detection. Analysis was performed by loading various amounts of a FITC-BSA conjugate per well onto a 4–20% Tris-glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% milk in TBST for at least 1 hour at room temperature. FITC-BSA was detected at ~72 kD using an FITC Monoclonal Antibody (#8) (Cat. No. MA5-14709) at a dilution of 1 µg/mL in 5% milk in TBST overnight at 4°C on a rocking platform, followed by a Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Cat. No. 31430) at a dilution of 1:50,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura Extended Duration Substrate (Cat. No.34076).