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Tytuł:
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Detection of Aspartic Proteinase Activities Using Gel Zymography.
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Autorzy:
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Perera HKI; Department of Biochemistry, Faculty of Medicine, University of Peradeniya, Galaha Rd, Peradeniya, Sri Lanka. .
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Źródło:
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Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2017; Vol. 1626, pp. 43-52.
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Typ publikacji:
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Journal Article
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Język:
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English
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Imprint Name(s):
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Publication: Totowa, NJ : Humana Press
Original Publication: Clifton, N.J. : Humana Press,
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MeSH Terms:
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Aspartic Acid Proteases/*analysis
Enzyme Assays/*methods
Native Polyacrylamide Gel Electrophoresis/*methods
Amido Black/analysis ; Animals ; Aspartic Acid Proteases/metabolism ; Cattle ; Coloring Agents/analysis ; Female ; Hemoglobins/chemistry ; Ovary/enzymology ; Staining and Labeling/methods ; Swine
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Contributed Indexing:
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Keywords: Aspartic proteinases; Hemoglobin; PAGE; Zymography
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Substance Nomenclature:
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0 (Coloring Agents)
0 (Hemoglobins)
EC 3.4.- (Aspartic Acid Proteases)
SZT789770M (Amido Black)
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Entry Date(s):
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Date Created: 20170614 Date Completed: 20180319 Latest Revision: 20180706
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Update Code:
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20240104
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DOI:
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10.1007/978-1-4939-7111-4_5
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PMID:
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28608199
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Gel zymography is a two-stage process where the proteins from the test sample are first separated by electrophoresis followed by the detection of the activity of hydrolytic enzymes. Many zymography procedures use sodium dodecyl sulfate (SDS) polyacrylamide gels copolymerized with an appropriate substrate. The procedure described here uses native polyacrylamide gel electrophoresis (PAGE) in the absence of both SDS and substrate. In order to visualize aspartic proteinase activity, the gel is impregnated in bovine hemoglobin at pH 3.0 for 15 min after the electrophoresis procedure. Subsequently, the gel is incubated in a humid container in the absence of hemoglobin for 1 h at 37 °C. At the end, the gel is stained with amido black and destained. Clear areas against a dark background corresponding to aspartic proteinase activities can be detected.