AAT Bioquest Sulfo-SMCC is a popular, water-soluble, amine-to-sulfhydryl crosslinker that contains NHS-ester and maleimide reactive groups at opposite ends of a medium-length cyclohexane spacer arm (8.3 Å).

Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) is a water-soluble, non-cleavable and membrane impermeable crosslinker. It contains an amine-reactive N-hydroxysuccinimide (NHS ester) and a sulfhydryl-reactive maleimide group. NHS esters react with primary amines at pH 7-9 to form stable amide bonds. Maleimides react with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds. The maleimide groups of Sulfo-SMCC and SMCC are unusually stable up to pH 7.5 because of the cyclohexane bridge in the spacer arm. Because it contains the hydrophilic sulfonyl moiety, Sulfo-SMCC is soluble up to ~10 mM in water and many commonly used buffers, thus avoiding the use of organic solvents which may perturb protein structure.

Sulfo-SMCC Highlights

• Amine reactive Sulfo-NHS ester crosslinks rapidly with primary amine-containing molecule
• Sulfhydryl-reactive maleimide reacts with cysteine residues to yield specific conjugates
• Sulfo-SMCC is water soluble, so crosslinking can be done in physiologic solutions
• High purity, crystalline Sulfo-SMCC can be used to create high-purity maleimide-activated derivatives
• Cyclohexane bridge confers added stability to the maleimide group making Sulfo-SMCC the ideal crosslinking agent for maleimide activation of proteins. Maleimide groups are stable for 64 hours in 0.1 M sodium phosphate buffer, pH 7 at 4°C.

Applications for Sulfo-SMCC

• Ideal crosslinker for enzyme labeling of antibodies, both enzyme activity and antibody specificity can be preserved
• Create specific bioconjugates via one- or two-step crosslinking reactions
• Create sulfhydryl-reactive, maleimide-activated carrier proteins for coupling haptens

Sulfo-SMCC is often used for the preparation of antibody-enzyme and hapten-carrier conjugates. In this type of conjugation, the NHS ester is reacted first with the antibody, excess crosslinking reagent removed and then the sulfhydryl-containing enzyme molecule is added. This two-step reaction scheme results in formation of specific antibody-enzyme conjugates.

Sulfo-SMCC is the reagent of choice for creating stable maleimide-activated carrier proteins that will spontaneously react with sulfhydryls. In this application, the NHS ester of Sulfo-SMCC is reacted with lysine residues on the carrier protein, converting them to reactive maleimides. These relatively stable maleimide-activated intermediates may be lyophilized and stored for later conjugation to a hapten.