Method for Determining Gelatinolytic Activity in Tissue Extracts: Real-Time Gelatin Zymography

This is a post-peer-review, pre-copyedit version of an article published in Methods in Molecular Biology, vol 1952. The final authenticated version is available online at: https://doi.org/10.1007/978-1-4939-9133-4_16. To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present a protocol for real-time gelatin zymography that is very useful for the detection of gelatin-degrading proteases in tissue extracts. This method uses fluorescence-labeled gelatin and therefore we also present an easy, fast, and cheap method for labeling gelatin with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF)