A new staining method for the detection of activities of H2O2-producing oxidases on gels and blots using cerium and 3,3'-diaminobenzidine.
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.
Seitz
J
J
Department of Anatomy and Cell Biology, Philipps University, Marburg, Germany.
Keppler
C
C
Fahimi
H D
HD
Völkl
A
A
D015100:3,3'-Diaminobenzidine; D000818:Animals; D002563:Cerium; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000378:metabolism*; D015151:Immunoblotting; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism*; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D013552:Swine
D015100:3,3'-Diaminobenzidine; D002563:Cerium; D006861:Hydrogen Peroxide; D010088:Oxidoreductases