Antibody-FITC Conjugation Protocol
Principle
Fluorescein isothiocyanate (FITC) is a widely used due to its high quantum efficiency and stability when conjugated. FITC exhibits a yellow-orange hue, characterized by its absorption peak at 495 nm. Upon excitation, it emits a yellow-green fluorescence, with its emission peak centered at 525 nm. The conjugation of FITC to antibodies represents an invaluable technique, offering the capacity to permanently tag biomolecules with a distinctive detectable property. This feature serves as a powerful tool for the visualization and tracking of the conjugate's interactions with other biomolecules. FITC's application extends to the identification of surface molecules, facilitating investigations through fluorescence microscopy or flow cytometry. Presented below is a detailed procedure outlining the coupling of antibody molecules with fluorescein derivatives.
Structure
FITC, derived from the nature fluorescein molecule, is characterized by the introduction of an isothiocyanate reactive group (−N=C=S), which replaces a hydrogen atom located on the lower ring of its molecular structure. The synthesis of FITC primarily involves the modification of the lower ring, specifically at the 5-carbon positions. This unique isothiocyanate functionality within FITC enables it to interact with primary amine groups found in proteins or antibodies, rendering it a pivotal tool for biomolecular labeling purposes.
Fig. 1 The molecular architecture of FITC.
Materials
- 1 to 2 mg/mL purified antibody
- 5 mg/mL FITC (Prepare the solution fresh)
- 0.2 M Sodium carbonate-bicarbonate buffer (pH 9.0)
- Phosphate buffered saline (PBS) buffer
- Amicon Ultra-0.5mL (MW= 3,000 Da)
- Sephadex G-25 column
Methods
Fig. 2 The pipeline of labeling antibody with FITC.
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Application
Fig. 3 Microscopic visualization of FITC-conjugated anti-SAG1 IgY antibody-stained T. gondii tachyzoites.1
This investigation marks a pioneering achievement in the development of a FITC-conjugated anti-SAG1 IgY antibody, as substantiated by ELISA, fluorescence spectrometry, and cell culture fluorescence imaging. The FITC-labeled IgY antibodies specific to T. gondii tachyzoites, thus obtained, hold promise for future deployment as diagnostic instruments for detecting T. gondii infections across diverse sample types.
Creative Biolabs offers antibody labeling services using FITC. For additional details, feel free to contact us without hesitation.
Reference
- Sert, Mehtap, Rabia Cakir Koc, and Yasemin Budama Kilinc. "Novel Fitc-labeled Igy antibody: fluorescence imaging Toxoplasma gondii in vitro." Scientific Reports 7.1 (2017): 852. Distributed under Open Access license CC BY 4.0, without modification.
