Staining Electrophoretic Gels for Laccase and Peroxidase Activity Using 1,8-Diaminonaphthalene

A new chromogenic substrate for laccases and peroxidases, 1,8-diaminonapthalene, was used to detect phenoloxidase activity in gels after SDS–PAGE. This substrate has several advantages over other widely used phenoloxidase stains in that it is inexpensive, and the oxidized product has both high molar...

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Bibliographic Details
Published in:Analytical biochemistry 2001-06, Vol.293 (1), p.96-101
Main Authors: Hoopes, James Todd, Dean, Jeffrey F.D.
Format: Article
Language:English
Subjects:
2-Naphthylamine - analogs & derivatives
2-Naphthylamine - chemistry
densitometry
electrophoresis
Electrophoresis, Polyacrylamide Gel
histochemistry
Immunoenzyme Techniques
Laccase
oxidase
Oxidation-Reduction
Oxidoreductases - analysis
peroxidase
Peroxidase - analysis
plant
Sensitivity and Specificity
Staining and Labeling - methods
zymogram
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Summary:A new chromogenic substrate for laccases and peroxidases, 1,8-diaminonapthalene, was used to detect phenoloxidase activity in gels after SDS–PAGE. This substrate has several advantages over other widely used phenoloxidase stains in that it is inexpensive, and the oxidized product has both high molar absorptivity and very low solubility. Furthermore, neither the substrate nor the product is known to have toxicity problems of the type associated with many other phenoloxidase stains. The sensitivity of detection using 1,8-diaminonapthalene was comparable to that obtained using the most sensitive stains commonly used for phenoloxidases, e.g., 3,3-diaminobenzidine, and was close to that attainable for protein detection using silver staining. Zymograms developed with 1,8-diaminonapthalene can be used with video densitometry to monitor the specific enzymatic activity of phenoloxidases during enzyme purification.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2001.5112