Analysis Note

Procedure1. Dissolve protein and FITC in carbonate-bicarbonate buffer.2. Slowly add FITC to protein with stirring. Cover with foil and stir 2 hours at room temperature.3. Separate conjugate from free FITC on G-25 column. Collect fractions.4. Pool fractions containing conjugate.5. Determine F/P ratio of conjugate spectrophotometrically.6. Stabilize with 1% bovine serum albumin and 0.1% sodium azide and store at 0-5 °C.

Application

The Flourotag FITC Conjugation Kit can be used for Immunohistochemisty and Immunocytochemistry using Flow Cytometry. It is also be used for the conjugation of FITC to peptide hormones, cytokines, growth factors and proteins.

Features and Benefits

Suitable for both small (1 mg) and large (5 mg) scale conjugations Completely aqueous procedure - no DMF needed Fast gel filtration separation of conjugate from excess FITC Complete protocols for conjugation and F/P ratio determination Sufficient reagents for at least 5 conjugations of 5 mg protein each and for optimization of F/P ratio before scale-up References for applications and protocols

Legal Information

FluoroTag is a trademark of Sigma-Aldrich Co. LLC

Principle

Sigma offers a convenient kit for preparing FITC-labeled antibodies. Fluorescein isothiocyanate (FITC), Isomer 1, is a widely used fluorophore, popular because of 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 free amino groups of proteins or peptides, forming a stable thiourea bond (see reaction). FITC conjugates of antibodies, lectins, hormones, and growth factors have been used in a variety of immunohistochemical and flow cytometry applications. The protocols have been optimized for antibodies, but may be adapted to other proteins by the end user.