Reaction Conditions for Chemical Coupling (S9236)

We recommend that coupling reactions between BC-NH2 and NHS esters take place in anhydrous N,N-dimethylformamide (DMF) using triethylamine as a base.

Coupling reactions typically use an excess of the BC-NH2 (1.5 equivalents) relative to the desired NHS label (1 equivalent) at 5-20 mM final concentration. Add a 1.5-fold molar excess of triethylamine in DMF. The reaction in generally performed at 30°C overnight.

The purification strategy will depend on the label used. Good results have been obtained with both HPLC and silica gel chromatography.

Example Reaction: Coupling of BC-NH2 to Alexa Fluor® 350 NHS-ester:

Coupling of BC-NH to Alexa Fluor® 350 NHS ester. A solution of 2.4 mg (5.8 µmol) Alexa 350 NHS-ester in 500 µl DMF was added to 2.0 mg (8.7 µmol)in 500 µl DMF. The reaction was shaken at 30°C overnight. The solvent was evaporated under vacuum. The reaction was diluted in 1 ml water / acetonitrile (9:1) and the product purified by reverse phase HPLC.