酶谱分析基础：在生物样品研究中的应用

Fundamentals of Zymography: Applications to the Study of Biological Samples.

作者信息

Recio-Aldavero Jorge, Muñoz-Moreno Laura, Román I Dolores, Bajo Ana M

机构信息

Research group: Cancers of Epithelial Origin. Facultad de Medicina y Ciencias de la Salud, Departamento de Biología de Sistemas, Unidad de Bioquímica y Biología Molecular, Campus Científico-Tecnológico, Universidad de Alcalá, Alcalá de Henares, 28805, Spain.

出版信息

Methods Mol Biol. 2025;2918:37-46. doi: 10.1007/978-1-0716-4482-9_4.

DOI:10.1007/978-1-0716-4482-9_4

PMID:40261612

Abstract

Zymography is a very useful technique to determine the gelatinolytic activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). This work describes the preparation and use of different biological samples (culture media, cell lysates, tissue extracts, and extracellular vesicles), as well as the determination of gelatinolytic activity in these samples. Proteins are separated on sodium dodecyl sulfate (SDS) polyacrylamide gels in the presence of gelatin. Once the total proteins have been resolved, specific activation of MMP-2 and MMP-9 found in the gel and staining-destination are performed to visualize this enzymatic activity. With a fast and simple method, it is possible to determine the catalytic activity of gelatinases 2 and 9 from different types of biological samples.

摘要

酶谱法是一种用于测定基质金属蛋白酶2和9（MMP - 2和MMP - 9）明胶酶解活性的非常有用的技术。这项工作描述了不同生物样品（培养基、细胞裂解物、组织提取物和细胞外囊泡）的制备和使用，以及这些样品中明胶酶解活性的测定。蛋白质在含有明胶的十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶上进行分离。一旦总蛋白质得到分离，就对凝胶中发现的MMP - 2和MMP - 9进行特异性激活以及染色显色，以可视化这种酶活性。通过一种快速简单的方法，可以测定来自不同类型生物样品的明胶酶2和9的催化活性。