Chapter 5 - Heterobifunctional Crosslinkers

Abstract

Heterobifunctional conjugation reagents contain two different reactive groups that can couple to two different functional targets on proteins and other macromolecules. The result is the ability to direct the cross-linking reaction to selected parts of target molecules, thus garnering better control over the conjugation process. Heterobifunctional reagents can be used to crosslink proteins and other molecules in a two- or three-step process that limits the degree of polymerization often obtained using homobifunctional crosslinkers. In a typical conjugation scheme, one protein is modified with a heterobifunctional compound using the crosslinker's most reactive or most labile end. The modified protein then is purified from excess reagent by gel filtration or rapid dialysis. Most heterobifunctionals contain at least one reactive group that displays extended stability in aqueous environments, therefore allowing purification of an activated intermediate before adding the second molecule to be conjugated.