E-64 is an irreversible and highly selective cysteine (thiol) protease inhibitor during isolation and purification of proteins and enzymes. It inhibits calpain activation.The inhibition of thiol proteases by E-64 appears to be of a non-competitive nature between the SH components. The trans-epoxysuccinyl group (active moiety) of E-64 irreversibly binds to an active thiol group in many cysteine proteases, such as papain, actinidase, and cathepsins B, H, and L to form a thioether linkage. E-64 does not inhibit serine proteases (except trypsin) like other cysteine protease inhibitors. It does not react with the functional thiol group of non-protease enzymes. E-64 can be used as a ligand for affinity purification of cysteine proteases. Although binding of the inhibitor is no longer irreversible, the specificity is retained. Effects on metastasis formation in mice (5).Stock solution: 1 mM aqueous solutionWorking solution: 1 to10 µMAqueous stock solutions are stable for months at -20 °C. Diluted solutions are stable for days at neutral pH.E-64 is also soluble in DMSO, a 10 mM solution can be prepared in dry DMSO and stored at -20 °C. Dilutions can be made in culture medium or in 0.9 % sodium chloride for injection. E-64 is an irreversible and highly selective cysteine (thiol) protease inhibitor during isolation and purification of proteins and enzymes. It inhibits calpain activation.The inhibition of thiol proteases by E-64 appears to be of a non-competitive nature between the SH components. The trans-epoxysuccinyl group (active moiety) of E-64 irreversibly binds to an active thiol group in many cysteine proteases, such as papain, actinidase, and cathepsins B, H, and L to form a thioether linkage. E-64 does not inhibit serine proteases (except trypsin) like other cysteine protease inhibitors. It does not react with the functional thiol group of non-protease enzymes. E-64 can be used as a ligand for affinity purification of cysteine proteases. Although binding of the inhibitor is no longer irreversible, the specificity is retained. Effects on metastasis formation in mice (5).Stock solution: 1 mM aqueous solutionWorking solution: 1 to10 µMAqueous stock solutions are stable for months at -20 °C. Diluted solutions are stable for days at neutral pH.E-64 is also soluble in DMSO, a 10 mM solution can be prepared in dry DMSO and stored at -20 °C. Dilutions can be made in culture medium or in 0.9 % sodium chloride for injection.