Description
Product Usage Information
Western Blotting: 1:1000
Flow Cytometry (Fixed/Permeabilized): 1:50 - 1:200
Storage
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at -20°C. Do not aliquot the antibody.
Protocol
Available protocols: Western Blotting, Flow Cytometry (Fixed/Permeabilized)
Specificity / Sensitivity
Carbamidomethyl Cysteine (F3T7J) Rabbit Monoclonal Antibody recognizes a broad range of carbamidomethylated cysteine containing proteins and peptides. This antibody does not cross-react with cysteines alkylated with desthiobiotin iodoacetamide (DBIA).
Species Reactivity: All Species Expected
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic peptide containing carbamidomethylated cysteine flanked by degenerate amino acids at positions N- and C-terminal to the modified cysteine.
Background
Cysteine alkylation is often performed with iodoacetamide (IAA) or chloroacetamide (CAA) to generate a carbamidomethyl (CAM) modification. Because alkylation occurs on reduced cysteines, disulfide-bonded cysteines must first be treated with dithiothreitol (DTT) or another reducing agent. The alkylation reaction is irreversible. Consequently, alkylated cysteines are unable to re-associate with each other. If the sulfhydryl group of a cysteine is already modified by another compound, that cysteine will not be able to react with IAA to form the carbamidomethyl modification. The alkylation reaction is rapid, although the efficiency can be influenced by the denaturant type used (1). Proteins are often derivatized to prevent disulfide bond scrambling that can lead to artifactual bands in gel electrophoresis, and to minimize protease inaccessibility of cysteine-containing regions that can limit coverage in mass spectrometry-based proteomics. In light of various studies describing potential non-cysteine reactivities of IAA and CAA and re-evaluating the effectiveness of such reactions, users should consider the benefits of cysteine derivatizations depending on their specific experimental goals (2-4).
