Fig 1. Schematic representation of a BCN-azide ligation reaction

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Condition1: EtOH/ H2O (3:2), room temperature
Condition2: DMSO/H2O, room temperature

BCN reagent is a class of click chemistry labeling reagents. BCN group can exclusively react with azide-tagged molecules or biomolecules to form a stable triazole. The click chemistry is also known as strain promoted alkyne-azide cycloaddition (SPAAC), BCN reagent has become widely used in bioconjugation, labeling and chemical biology.

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BCN click chemistry can be run in aqueous buffer or in organic solvents depending on the property of the substrate molecules.Reagents with PEG arm will increases the compound's hydrophilicity. 

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This method requires to activate the biomolecule #1 with BCN reagent, and the biomolecule #2 with azide, then to mixing the two activated biomolecules to form a conjugate.

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Features

• Biocompatibility – no cytotoxic Copper catalyst required – Nice of in-vivo applications.

• Mild conditions – conjugation in aqueous buffered media and at low temperature • Stability – BCN and azide moieties are long term stable

• Efficiency – formation of a stable triazole in quantitative yield

• Specificity and Bioorthogonality azide react only with BCN in the presence of -NH2, --COOH and other protein functional group;

Note: Thiol will react BCN, and β-mcercaptoethanol (β-ME) will suppress this thiol-BCN addition.

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Example protocol: (Antibody-oligo conjugation)
Antibody activation
• Mix antibody with 20-30-fold molar excess of BCN NHS ester dissolved in DMSO (10 mM solution). DMSO content in the final mixture should be around 20%, antibody concentration in the reaction mixture around 1 mg/mL.
• Incubate at room temperature for 60 minutes.
• Add Tris (10 uL, 100mM in water) to the reaction to quench the unreacted DBCO-NHS ester.
• Incubate for 15 minutes.
• Remove the unreacted BCN-NHS ester using spin desalting column.
Note: BCN-functionalized antibody can be stored at -20°C for up to months.

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Conjugation

• Mix BCN-functionalized antibody or another biomolecule with 2-4x molar excess of azide modified oligonucleotide or azide functionalized dye.

• Incubated overnight at room temperature.

• Validate your final conjugate using SDS gel electrophoresis

• Remove the unreacted oligonucleotide or dye using liquid chromatography (reverse phase HPLC, ion exchange HPLC, or both).

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