Abstract
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.
Highlights
Matrix metalloproteinases (MMPs) are Ca2+/Zn2+ dependent endopeptidases necessary for the degradation of extracellular matrix (ECM) components during a multitude of physiological and pathophysiological processes [1,2,3]
SDS gels copolymerized with a suitable protein substrate are used and protease activities can be analyzed with this method after enzyme renaturation through SDS-removal following electrophoretic separation [8]
Additional benefits of this method include (a) the possibility to detect and quantify several proteases exerting activity on the same substrate in parallel, (b) detection of the proteolytically inactive pro-forms of proteinases since these become activated during the renaturation procedure, and (c) removal of tissue inhibitors of metalloproteinases (TIMPs) during electrophoresis allows for the detection of the total enzymatic activity in a given sample
Summary
Matrix metalloproteinases (MMPs) are Ca2+/Zn2+ dependent endopeptidases necessary for the degradation of extracellular matrix (ECM) components during a multitude of physiological and pathophysiological processes (e.g., embryonic development, aging, tissue remodeling, tumor growth, and invasion) [1,2,3]. SDS gels copolymerized with a suitable protein substrate (e.g., gelatin) are used and protease activities can be analyzed with this method after enzyme renaturation through SDS-removal following electrophoretic separation [8] Additional benefits of this method include (a) the possibility to detect and quantify several proteases exerting activity on the same substrate in parallel, (b) detection of the proteolytically inactive pro-forms of proteinases since these become activated during the renaturation procedure, and (c) removal of tissue inhibitors of metalloproteinases (TIMPs) during electrophoresis allows for the detection of the total enzymatic activity in a given sample. The standard staining procedure for protein gels with Coomassie is well established and remained largely unchanged for almost 30 years [9,10,11] This is a very sensitive and robust technique, it requires considerable amounts of ethanol and acetic acid during the subsequent destaining steps, a fact that can substantially contribute to the final costs for analyzing zymogram gels, especially in high throughput settings. Using the method presented in this manuscript one can roughly estimate that replacing the Coomassie staining procedure with the Ponceau staining protocol can further reduce the costs of gel destaining by more than 70% from € 6.14 to € 1.71 by using the volumes indicated in the Procedure section
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