5-FITC-Tetrazine is an fluorescent dye composed of FITC and Tetrazine (Ex/Em = 494 nm/520 nm). 5-FITC-Tetrazine can be used for fluorescent labeling and imaging [1].
In Vitro, Guide (The following is our recommended solution. This solution merely provides guidance; it should be modified according to your specific needs.) 1. Reagent Preparation 1.1 Reconstitution/Preparation: Dissolve 5-FITC-Tetrazine in anhydrous DMSO or DMF to prepare a 1-10 mM stock solution. 1.2 Storage: Store the dry powder and reserve solution at -20°C in a dark place. Pay attention to avoiding light and moisture to prevent the degradation of the tetrazine and fluorescent core. 1.3 Buffer: Use PBS or compatible neutral physiological aqueous buffer (pH 7.2 - 7.4). If the previous steps involve free isothiocyanate reactive activity, avoid using buffers containing primary amines (such as Tris or glycine). 2. Labelling Procedure 2.2 Target Preparation: Ensure that your protein, antibody, or cell surface target has been labeled with a strained diene acceptor tag (such as TCO). 2.3 Incubation/Loading: Add the 5-FITC-Tetrazine stock solution to the TCO-labeled biological molecule solution (usually using a 2 to 5-fold molar excess of the dye). 2.4 Reaction conditions: Incubate at room temperature for 5 to 30 minutes (or up to 1 hour at 4°C). The IEDDA reaction can proceed rapidly without the need for any toxic metal catalyst. 2.5 Purification: Remove the unreacted free dyes through size exclusion chromatography (centrifugal column) or by thorough dialysis in PBS.
References:
[1]. Longo B, et al. Design, Synthesis, Conjugation, and Reactivity of Novel trans,trans-1,5-Cyclooctadiene-Derived Bioorthogonal Linkers. Bioconjug Chem. 2020 Sep 16;31(9):2201-2210.
In Vitro, Guide (The following is our recommended solution. This solution merely provides guidance; it should be modified according to your specific needs.) 1. Reagent Preparation 1.1 Reconstitution/Preparation: Dissolve 5-FITC-Tetrazine in anhydrous DMSO or DMF to prepare a 1-10 mM stock solution. 1.2 Storage: Store the dry powder and reserve solution at -20°C in a dark place. Pay attention to avoiding light and moisture to prevent the degradation of the tetrazine and fluorescent core. 1.3 Buffer: Use PBS or compatible neutral physiological aqueous buffer (pH 7.2 - 7.4). If the previous steps involve free isothiocyanate reactive activity, avoid using buffers containing primary amines (such as Tris or glycine). 2. Labelling Procedure 2.2 Target Preparation: Ensure that your protein, antibody, or cell surface target has been labeled with a strained diene acceptor tag (such as TCO). 2.3 Incubation/Loading: Add the 5-FITC-Tetrazine stock solution to the TCO-labeled biological molecule solution (usually using a 2 to 5-fold molar excess of the dye). 2.4 Reaction conditions: Incubate at room temperature for 5 to 30 minutes (or up to 1 hour at 4°C). The IEDDA reaction can proceed rapidly without the need for any toxic metal catalyst. 2.5 Purification: Remove the unreacted free dyes through size exclusion chromatography (centrifugal column) or by thorough dialysis in PBS.
References:
[1]. Longo B, et al. Design, Synthesis, Conjugation, and Reactivity of Novel trans,trans-1,5-Cyclooctadiene-Derived Bioorthogonal Linkers. Bioconjug Chem. 2020 Sep 16;31(9):2201-2210.