Kinase experiment:

The standard reaction mixture contains 1×l0-7 M MCLA hydrochloride (MCLA), 5×10-5 M hypoxanthine, XOD (6.5 U), SOD (0.2 to 20 ng/mL) or none, and 50 mM Tris-HCl buffer containing 0.1 mM EDTA at pH 7.8, in a total volume of 3.0 mL. Chemiluminescence measurement is initiated by the addition of MCLA hydrochloride to the standard incubation mixture excluding XOD, continued for 4 min without additive and for an additional 4 min after the addition of XOD. Chemiluminescence is measured using a luminescence reader at 25℃[1].

MCLA hydrochloride is a chemiluminescent reagent which can be used to quantify aqueous concentrations of superoxide.

The non-specific luminescence remains almost constant for 10 min after the addition of MCLA hydrochloride (MCLA) and is not significantly influenced by SOD. The MCLA hydrochloride method is, however, 4.5-times more sensitive than the CLA method[1].

[1]. Kimura H, et al. Highly sensitive and reliable chemiluminescence method for the assay of superoxide dismutase inhuman erythrocytes. FEBS Lett. 1988 Nov 7;239(2):347-50.