NHS (N-hydroxysuccinimide)

No. EDC is very unstable in aqueous environments and must be dissolved immediately before use. NHS and Sulfo-NHS are relatively stable in solution but best results are obtained when they are dissolved immediately before use. Store these compounds desiccated at 4°C.

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Yes. Except for the intended targets, reactions must not contain carboxyl or amine compounds. Thus, Tris, glycine, lysine, ethanolamine or other amine- containing buffers must be avoided.

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Sulfo-NHS is the sulfonate sodium salt of NHS; it is water-soluble, but not membrane-permeable. NHS is membrane-permeable and water-soluble. NHS is soluble in organic solvents, as is Sulfo-NHS to a lesser extent. Because NHS is a leaving group in reactions with primary amines, the final conjugation product resulting from NHS and Sulfo-NHS reactions is identical. Sulfo-NHS is chosen for its ability to confer better solubility to the activated compound and/or to control its membrane permeability.

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These compounds are used to modify a carboxyl group to form an amine-reactive ester. This is accomplished by mixing NHS with a carboxyl-containing molecule and the carbodiimide EDC (Cat. No. 22980, 22981, 77149, A35391). EDC causes a dehydration reaction between the carboxyl and the NHS hydroxyl group, giving rise to an NHS-ester-activated molecule. The activated molecule can then be reacted spontaneously with a primary amine-containing molecule. Although the carboxyl-molecules can be made to react directly with amines using EDC, the reaction is much more efficient with NHS because a stable intermediate is created. In fact, molecules that are activated as NHS esters can be dried and stored for later reaction to amine-containing targets.

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These compounds are used in conjunction with the crosslinker EDC (Cat. No. 22980, 22981, 77149, A35391) to synthesize amine-reactive labeling reagents, crosslinkers and conjugation compounds. Any compound containing a carboxylic acid (-COOH), such as a protein, or biotin or peptide, can be activated with NHS or Sulfo-NHS to form an NHS ester that will spontaneously react to form covalent amide linkages with proteins and other molecules that contain primary amines (-NH2).

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