Sulfhydryls conjugation with haptens

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this crosslinker to make antibody-enzyme or hapten-carrier conjugates may be found in Chapter 20, Section 1.1 and Chapter 19, Section 5, respectively. 

Figure 19.16 A common way of conjugating sulfhydryl-containing haptens to carrier proteins is to activate the carrier with sulfo-SMCC to create an intermediate maleimide derivative. The maleimide groups then can be coupled to thiols to form thioether bonds.

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). 

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