FluoroTag^™ FITC Conjugation Kit 

Procedure
1. 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.

• 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

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.

FluoroTag is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.

2-8°C

Sender, S., et al., Localization of carbonic anhydrase IV in rat and human heart muscle. J. Histochem. Cytochem. 46, 855-861, (1998) abstract

Mullins, R.F., et al., Characterization of drusen-associated glycoconjugates. Ophthalmology 104, 288-294, (1997) abstract

Blackmore P.F., Extragenomic actions of progesterone in human sperm and progesterone metabolites in human platelets. Steroids 64, 149-156, (1999) abstract

Lloyd-Evans, P., et al., Use of a phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry. Transfus. Med. 9, 155-160, (1999) abstract

Hidaka, H., et al., The identification of specific high density lipoprotein3 binding sites on human blood monocytes using fluorescence-labeled ligand. J. Lipid Res. 40, 1131-1139, (1999) abstract

Kobayashi, Y., et al., Effects of a GnRH analogue on human smooth muscle cells cultured from normal myometrial and from uterine leiomyomal tissues. Mol. Hum. Reprod. 3, 91-99, (1997) abstract

S2002, Sodium azide

A9647, Albumin from bovine serum

0.1 M Carbonate-bicarbonate buffer capsule, pH 9.0 5 capsules

Fluorescein isothiocyanate isomer I, suitable for protein labeling, ≥90% (HPLC), powder 5×2 mg F7250

Phosphate buffered saline, powder, pH 7.4 5 packages P3813

Sephadex G-25 column, 3.5 mL 1

Sephadex G-25 column, 9.1 mL 1