Homobifunctional Sulfhydryl-Reactive Crosslinkers

The differences within these families of reagents generally relate to the length of the spacer or bridging portion of the molecule. Occasionally, the bridging portion itself is designed to be cleavable by one of a number of methods (Chapter 8). The great majority of homobifunctional, sulfhydryl-reactive crosslinkers mentioned in the literature are not readily available from commercial sources and would have to be synthesized to make use of them. The ones listed in this section are obtainable from Thermo Fisher. 

DPDPB is insoluble in aqueous solutions and should be initially dissolved in an organic solvent prior to addition of a small aliquot to a buffered reaction medium. Preparation of a stock solution in DMSO at a concentration of 25 mM DPDPB works well. The addition of an aliquot of this stock solution to the conjugation reaction should not result in more than about 10 percent organic solvent by volume in the buffered mixture or protein precipitation may occur. 

DPDPB has been used to study the endocytosis of cadherin from intracellular junctions (Troyanovsky et al., 2006), the subunit arrangement in the flagellar rotor assembly (Lowder et al., 2005), and the disease-associated mutations in myelin proteolipid protein in the endoplasmic reticulum (ER) (Swanton et al., 2005). DPDPB can be used to conjugate reduced antibody molecules to p-D-galactosidase using essentially the same protocol as that described by O Sullivan et al. (1979). 

B/s-maleimidohexane (BMH) is a homobifunctional reagent containing a non-cleavable, 6-atom spacer between terminal maleimides (Thermo Fisher). The maleimide groups can react 

Difluorobenzene derivatives are small homobifunctional crosslinkers that react with amine groups. Conjugation using these compounds results in bridges of only about 3 A in length, potentially providing information concerning very close interactions between macromolecules. 

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