MMP Activity Detection in Zymograms
MMP Activity Detection in Zymograms
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DOI：
10.1007/978-1-4939-7111-4_6
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发表时间：
2017-01-01
期刊：
影响因子：
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通讯作者：
Ferdinandy, Peter
中科院分区：
文献类型：
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作者：
Bencsik, Peter;Bartekova, Monika;Ferdinandy, Peter
基质金属蛋白酶（MMP）属于一类独特的锌依赖性内肽酶。酶谱法是一种半定量工具，用于测定各种生物样品中不同MMP同工酶的活性。在底物凝胶酶谱法中，不同来源的蛋白质样品（组织、细胞裂解物、血浆/血清、灌注液、其他液体）在含有共聚底物（明胶、酪蛋白、弹性蛋白等）的十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶中分离，在孵育后，基质金属蛋白酶能够切割底物，凝胶染色后检测到基质金属蛋白酶活性，呈深蓝色背景下的透明条带。原位酶谱法是冷冻切片中底物酶谱法的组织学修饰，允许检测组织内MMP活性的定位。在这里，我们描述了所有上述技术的详细实验方案，并提供了几个样品测量的例子。
Matrix metalloproteinases (MMP) belong to a distinguished class of zinc-dependent endopeptidases. Zymography is a semi-quantitative tool for determining the activity of different MMP isoenzymes in a variety of biological samples. In substrate gel zymography, protein samples of different origin (tissue, cell lysates, plasma/serum, perfusates, other liquids) are separated in sodium dodecyl sulfate (SDS) polyacrylamide gels containing copolymerized substrate (gelatin, casein, elastin, etc.), and after incubation-enabling substrate cleavage by MMPs, MMP activities are detected after the gel staining as transparent bands against a dark-blue background. In situ zymography is a histological modification of substrate zymography in frozen sections, allowing detection of the localization of the MMP activities within the tissue. Here, we describe detailed experimental protocols of all abovementioned techniques and provide examples for several sample measurements.