Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography

This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 1626; p. 33
Main Authors Kernaghan, Gavin, Mayerhofer, Michael
Format Journal Article
LanguageEnglish
Published United States 2017
Subjects
Animals
Aspartic Acid Proteases - analysis
Aspartic Acid Proteases - metabolism
Cattle
Cell Culture Techniques - methods
Culture Media - metabolism
Electrophoresis, Polyacrylamide Gel - methods
Enzyme Assays - methods
Fungal Proteins - analysis
Fungal Proteins - metabolism
Fungi - enzymology
Fungi - growth & development
Fungi - metabolism
Hydrogen-Ion Concentration
Serum Albumin, Bovine - metabolism
SDS-PAGE
Protein utilization
Laemmli gels
Fungal enzymes
Neutral zymography
Bradford assay
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Summary:This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.
ISSN:1940-6029
DOI:10.1007/978-1-4939-7111-4_4