Wechselberger Christian, Doppler Christian, Bernhard David
Center for Medical Research, Medical Faculty, Johannes Kepler University, 4020 Linz, Austria.
Methods Protoc. 2019 Jul 24;2(3):61. doi: 10.3390/mps2030061.
Zymography is a widely used electrophoretic method to determine proteolytic activities in samples from various sources. The method is based on copolymerizing a suitable protein substrate within a sodium dodecyl sulfate-polyacrylamide gel. Following electrophoretic separation of the protease containing samples and a suitable incubation period, degradation of the substrate can be visualized through staining with Coomassie blue. Sites of proteolysis become visible as white bands on a dark blue background. However, this staining protocol requires considerable amounts of ethanol and acetic acid to remove unbound dye molecules. In this report, we describe a new staining protocol using Ponceau S which offers substantial advantages in terms of assay usability and cost reduction, especially when performing large quantities of zymograms or in resource-limited settings. Fast and reproducible staining of zymograms with our protocol is demonstrated, and reliable quantitation of proteolytic activity in comparison to the standard Coomassie staining procedure is shown.
酶谱法是一种广泛应用的电泳方法，用于测定来自各种来源的样品中的蛋白水解活性。该方法基于在十二烷基硫酸钠-聚丙烯酰胺凝胶中共聚合适的蛋白质底物。在对含有蛋白酶的样品进行电泳分离并经过适当的孵育期后，通过考马斯亮蓝染色可以观察到底物的降解情况。蛋白水解位点在深蓝色背景上呈现为白色条带。然而，这种染色方案需要大量的乙醇和乙酸来去除未结合的染料分子。在本报告中，我们描述了一种使用丽春红S的新染色方案，该方案在检测可用性和成本降低方面具有显著优势，特别是在进行大量酶谱分析或资源有限的情况下。我们的方案展示了对酶谱进行快速且可重复的染色，并与标准考马斯染色程序相比，显示了对蛋白水解活性的可靠定量。
