EZ-Link™ DBCO Protein Labeling Kit (Thermo Scientific™)

The EZ-Link DBCO Protein Labeling Kit provides everything needed to covalently label proteins with a dibenzylcyclooctyne (DBCO) group for copper-free click chemistry applications. This kit contains the necessary materials to perform five labeling reactions and subsequent purification for removal of excess DBCO reagent. The TFP-PEG4-DBCO provided in this kit readily reacts with the primary amines found on proteins to form a covalently attached DBCO group that can be further reacted with an azide-labeled coupling partner (supplied separately) to create diverse bioconjugates.

EZ-Link DBCO Protein Labeling Kit features include:
• Versatile—label proteins (20–150 kDa) with volumes of 0.5–5 mL
• Chemoselective—DBCO reacts selectively with azides
• Heterobifunctional—reactive towards amine and azides
• Stable—TFP esters are more hydrolytically stable than NHS esters enabling less reagent to be used
• Convenient—labeling and purification can be completed in as little as 90 minutes with 30 min hands-on time

The TFP-PEG4-DBCO provided in this kit is a heterobifunctional crosslinker that contains an amine-reactive tetrafluorophenyl (TFP) group and an azide-reactive dibenzylcyclooctyne (DBCO) group with a water-solubilizing PEG4 spacer in between. In the first conjugation step, the TFP group reacts with a primary amine on a protein to form a stable amide bond and a covalently bound DBCO moiety. Subsequent purification removes excess crosslinker from solution that may interfere with the second labeling step. Once a protein is DBCO-labeled and purified, it can undergo a copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) to form a stable triazole linkage with an azide-labeled coupling partner (supplied separately) for the creation of diverse bioconjugates.

Dibenzylcyclooctynes (DBCO) and azides are bioorthogonal coupling partners as they can react in a biological system without interfering with normal biochemical processes, and as a result there is minimal off-target labeling of macromolecules found in cells or lysates. The reaction between a DBCO and azide can be used for a variety of applications including creation of protein-protein, protein-biomolecule, or protein-small molecule conjugates.
Classic click reactions involve a copper-catalyzed azide-alkyne cyclo­addition to label or conjugate molecules. A drawback of this approach is that copper ions can impair protein function, harm cells, and reduce the fluorescence of fluorophores. DBCO is distinct in that it doesn’t require copper to catalyze the reaction with an azide for triazole formation. The strain in the eight-membered ring allows the reaction with azide-modified molecules to occur in the absence of catalysts, preventing copper-induced damage to cells or proteins.