FITC can bind to various antibody proteins, and the bound antibody does not lose the specificity of binding to a certain antigen, and has strong yellow-green fluorescence in alkaline solution. The corresponding antigen can be qualitatively, localized or quantitatively detected by observation under a fluorescence microscope or flow cytometry analysis. Used in medicine, agriculture and animal husbandry, it can quickly diagnose diseases caused by bacteria, viruses and parasites.
FITC labeled antibody process
(1) The protein to be crosslinked (concentration ≥1mg/mL) was dialyzed three times against the crosslinking reaction solution at 4°C to pH=9.0. The preparation method of the crosslinking reaction solution: 7.56g NaHCO3, 1.06g Na2CO3, 7.36g NaCl, add water to make the volume to 1 L.
(2) Dissolve FITC in DMSO at a concentration of 1 mg/mL. FITC used for each cross-linking should be freshly prepared and protected from light.
(3) Slowly add FITC to the antibody solution at the ratio of P:F (Protein: FITC) = 1mg: 150μg, and gently shake to mix it with the antibody while adding, and react at 4°C in the dark for 8 hours.
(4) Add 5mol/L NH4Cl to a final concentration of 50mmol/L, and terminate the reaction at 4°C for 2h.
(5) Dialyze the cross-linked substance in PBS for more than four times until the dialysate is clear.
(6) Identification of cross-linked products
Protein concentration (mg/mL) = [A280– 0.31×A495] / 1.4
F/P ratio: 3.1×A495/ [A280 – 0.31×A495], the value should be between 2.5 and 6.5.
(7) FITC cross-linked protein should be placed in a pH 7.4 phosphate buffer, added with 0.1% NaN3, 1% BSA, and stored at 4°C in the dark.
Application：
6-Fluorescein Isothiocyanate is a fluorescent reagent used in fluorescence polarization binding assay to develop inhibitors of inactive p38α Mitogen-activated protein kinase (MAPK).
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