FluoroTag™ FITC Conjugation Kit
SIGMA/FITC1
Synonym: FITC
Product Type: Chemical
Conjugation Reaction.
Fluorescent micrograph of mitotic newt lung cells. Microtubules are stained with monoclonal anti-β-tubulin and Goat Anti-Mouse IgG FITC conjugate (F 0257). Chromosomes are counterstained with Hoechst 33342. The astral microtubules are green, the spindle is yellow, and the chromosomes are blue. [From C. Rieder, Wadsworth Center, Division of Molecular Medicine, Lab of Cell Regulation, Albany, NY].
| fluorescence | λex 495 nm; λem 525 nm |
| packaging | pkg of (Kit contains reagents for 5 conjugations.) |
| Quality Level | 200  |
| storage temp. | 2-8°C |
| Analysis Note: | 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. |
| 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. |
| Biochem/physiol Actions: | 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. |
| 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 |
| Symbol |  GHS07 |
| Signal word | Warning |
| Hazard statements | H319 |
| Precautionary statements | P305 + P351 + P338 |
| Hazard Codes | Xi |
| Risk Statements | 36 |
| Safety Statements | 26 |
| RIDADR | NONH for all modes of transport |
| Flash Point(F) | Not applicable |
| Flash Point(C) | Not applicable |
| Storage Temp. | 2-8°C |
| UNSPSC | 12352200 |
| Components | Phosphate buffered saline, powder, pH 7.4, for preparing 1 L solutions 5 pkg; Sephadex G-25 column, 3.5 mL 1; Sephadex G-25 column, 9.1 mL 1; Fluorescein isothiocyanate isomer I - F7250KC-2MG 2 mg |