A new staining method for the detection of activities of H₂O₂‐producing oxidases on gels and blots using cerium and 3,3′‐diaminobenzidine

Abstract

Cerium chloride (CeCl3) was used to trap the hydrogen peroxide generated by several oxidases on native gels and blots. The pale yellow color of cerium perhydroxide formed is converted to a brown-black precipitate by the subsequent reaction with 3,3'-diaminobenzidine. The suitability of this method for the detection of the activity of several oxidases on gels and on blots under nondenaturing conditions, employing different electrophoretic systems and resolving techniques, is demonstrated. Moreover, this method has proven to be highly suitable for the assessment of the substrate and stereospecificity of oxidases, the determination of their molecular weights, and the isoelectric points of isoforms.

Differentiation-specific localization of catalase and hydrogen peroxide, and their alterations in rat skin exposed to ultraviolet B rays

“…Statistical analysis was performed using the unpaired Student's t-test. *p < 0.05 was considered to be significant *p < 0.05, **p < 0.01. formed an in situ H 2 O 2 assay using the cerium-DAB method [8]. In situ H 2 O 2 assay has previously been used to detect the generation of H 2 O 2 by leukocytes infiltrating the epidermis upon exposure to UVB in adult mice [19] and human skin [20].…”

“…This enzyme catalyzes the transfer of electrons and hydrogen to oxygen, thus this oxidizing reaction generates H 2 O 2 in the upper epidermis [5,7,8].…”

Rapid Activation of Glutamate Cysteine Ligase following Oxidative Stress

“…Reactions were initiated with fresh assay buffer containing 15 mM glutamate, 5 mM cysteine, and 3 mM cerium chloride (CeCl 3 ), and the gels were incubated at 37°C for 30 -60 min with shaking. The in-gel activity assay exploits the deposition of CePO 4 crystals following ATP hydrolysis (18,19). CePO 4 crystals were visualized on a Gel-doc video capture system (BioRad) using an MVI Darklite trans-illuminating light source (Meridian, Kent, WA) adapted to orthogonally illuminate the gel.…”