A new protein crosslinking method for labeling and modifying antibodies (TECH2P.758)

The modification and labeling of a protein with a tag macromolecule is an important task in biological research. There are a few methods available for crosslinking two macromolecules. The direct crosslinking of two macromolecules suffer extremely low yield and difficult purification. A common method is to use a small crosslinker (such as SMCC) in numerous bioconjugations, but SMCC-modified protein is extremely unstable and often self-reactive during storage since proteins often contain both amine and thiol groups that cause significant amount of homo-crosslinking. We have recently developed an effective crosslinking method to selectively link two macromolecules. The new crosslinking method uses two unique crosslinkers that readily react upon mixing. Specifically, a pair of crosslinkers is used, including Buccutite FOL and Buccutite MTA. A protein (e.g., antibody) is first labeled with Buccutite FOL, and the tag macromolecule is labeled with Buccutite MTA. The two resulted Buccutite-modified conjugates can be readily purified by a desalting column or dialysis. Buccutite FOL-labeled protein and Buccutite MTA-tag macromolecule are mixed under extremely mild neutral conditions to give the desired protein-tag macromolecule conjugate. The Buccutite bioconjugation system is run at neutral pH, and completed within 1 hour. The conjugation can be run at low concentration, thus eliminating the pretreatment step for some diluted proteins. No catalyst is required.