Heterobifunctional cross-linkers

Weltman, J.K., Hohnson, S.-A., Langevin, J., and Riester, E.F. (1983) N-Succinimidyl(4-iodoacetyl)amino benzoate Anew heterobifunctional cross-linker. BioTech. 1, 148-152. 

Photoreactive Amino Acids and Heterobifunctional Cross-Linkers 181... 

The application of heterobifunctional cross-linkers allows macromolecular PAL to probe protein-protein interactions, including subunit interactions and location, monitoring the conformational changes induced by signal transmission. 

Purify the reduced IgG from excess 2-mercaptoethylamine and reaction byproducts by dialysis or gel filtration using Sephadex G-25. All buffers should contain 1—10 mM EDTA to preserve the free sulfhydryls from metal-catalyzed oxidation. The sulfhydryl containing half-antibody may now be used in conjugation protocols that use—SH-reactive heterobifunctional cross-linkers (Chapter 10, Section 1.1). 

Succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) is a heterobifunctional cross-linker with significant utility in cross-linking proteins, particularly in the preparation of antibody-enzyme and hapten-carrier conjugates (Hashida and Ishikawa, 1985 Dewey et al., 1987). The NHS ester end of the reagent can react with primary amine groups on proteins to form stable amide bonds. The maleimide end of SMCC is specific for coupling to sulfhydryls when the reaction pH is in the range of 6.5-7.5 (Smyth et al., 1964) (Fig. 158). 

N-Succinimidyl-4(4-azidophenyl)l,3 -dithiopropionate (SADP) is a photoreactive heterobifunctional cross-linker that is cleavable by treatment with a disulfide reducing agent (Pierce). The cross-linker contains an amine-reactive NHS ester and a photoac-tivatable phenylazide group, providing specific, directed coupling at one end and nonselective insertion at the other end. 

---