AF 488 azide

Method for Labeling Oligonucleotides with AF 488 Azide^[1]
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Reagent Preparation: Prepare 5'-alkyne-modified oligonucleotide; AF 488 azide solution in dimethyl sulfoxide (DMSO); ascorbic acid (dissolved in 2M triethylammonium acetate buffer); a premixed solution of CuSO₄·5H₂O and TBTA (Tris-(benzyltriazolylmethyl)amine; Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)Methyl]aMine) (dissolved in 55% DMSO aqueous solution); degassed deionized water; DMSO; acetone precipitation solution containing 2% LiClO₄; pure acetone.
2. Fluorescent Labeling Reaction: In a reaction tube, sequentially add alkyne-modified oligonucleotide (30nmol), degassed deionized water (60μL), DMSO (60μL), ascorbic acid solution (15μL), and AF 488 azide DMSO solution (5μL of 100mM). Mix the mixture thoroughly. Add the CuSO₄·5H₂O-TBTA premixed solution (6μL) to the reaction mixture. Incubate the sealed reaction tube at room temperature, protected from light, for 3 hours.
3. Product Purification: After the reaction, add pre-cooled 2% LiClO₄ acetone solution (1.5mL) to the mixture. Precipitate at -20°C for 30 minutes. Centrifuge at 10,000rcf to obtain the precipitate. Wash the precipitate with pure acetone. Dry to obtain the AF 488-labeled oligonucleotide.
Precautions:
(1) This method is based on the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, which is mild and suitable for oligonucleotide labeling in aqueous phases.
(2) The reaction should be performed protected from light to prevent quenching of the AF 488 azide fluorophore.
(3) For your safety and health, please wear a lab coat and disposable gloves during operation.