Method for Determining Gelatinolytic Activity in Tissue Extracts: Real-Time Gelatin Zymography

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we pr...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 1952; p. 201
Main Authors Hadler-Olsen, Elin, Winberg, Jan-Olof
Format Journal Article
LanguageEnglish
Published United States 2019
Subjects
Animals
Cell Line
Electrophoresis, Polyacrylamide Gel - methods
Enzyme Assays - methods
Fluorescent Dyes - analysis
Fluorescent Dyes - metabolism
Furans - analysis
Furans - metabolism
Gelatin - analysis
Gelatin - metabolism
Gelatinases - analysis
Gelatinases - metabolism
Humans
Kidney - chemistry
Kidney - enzymology
Liver - chemistry
Liver - enzymology
Mice
Mice, Inbred BALB C
Skin - chemistry
Skin - enzymology
MDPF-gelatin
Tissue
Fluorescence labeling of gelatin
Matrix metalloproteases
Proteases
Gelatinase
Real-time gelatin zymography
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Summary:To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present a protocol for real-time gelatin zymography that is very useful for the detection of gelatin-degrading proteases in tissue extracts. This method uses fluorescence-labeled gelatin and therefore we also present an easy, fast, and cheap method for labeling gelatin with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF).
ISSN:1940-6029
DOI:10.1007/978-1-4939-9133-4_16