Thermo Scientific Pierce N-Ethylmaleimide (NEM) is a small compound that forms stable, covalent thioether bonds with sulfhydryls (e.g., reduced cysteines), enabling them to be permanently blocked to prevent disulfide bond formation.
N-Ethylmaleimide (NEM) is an alkylating reagent that reacts with sulfhydryls. At pH 6.5-7.5, the maleimide reaction is specific for sulfhdryls; however, at pH values greater than 7.5, reactivity with amino groups occurs. Maleimide groups react with sulfhydryls by nucleophilic attack of the thiolate anion on one of the carbons of the double bond. When sufficient sulfhydryls have been blocked, the reaction can be monitored by measuring the decrease in absorbance at 300nm as the double bond becomes a single bond. The resulting thioether group is non-reversible and terminates in an ethyl group, blocking or capping the sulfhydryl.
- Permanently block sulfhydryls to prevent disulfide bond formation.
- Monitor the reaction by measuring the decrease in absorbance at 300nm
- Block sulfhydryl-containing reagents that interfere in enzyme assays
Structure and properties of NEM:
- Functional groups: maleimide (sulfhydryl-reactive)
- Chemical name: N-ethylmaleimide
- CAS number: 128-53-0
- Chemical formula: C6H7NO2
- Molecular weight: 125.13
- Form: white to off-white crystals or powder
- Partis, M.D., et al. (1983). Cross-linking of protein by ω-maleimido alkanoyl N-hydroxysuccinimido esters. J Prot Chem 2(3):263-77.
Overview of crosslinking and protein modification
Iodoacetamide – carboxymethylate sulfhydryls
MM(PEG)n – methylate and pegylate sulfhydryls
MMTS – reversibly block sulfhydryls