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

Abstract

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localise them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrade 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 labelled gelatin and therefore, we also present an easy, fast and cheap method for labelling gelatin with 2-Methoxy-2,4-Diphenyl-3(2H)-Furanone (MDPF).

Molecular Interactions Stabilizing the Promatrix Metalloprotease-9·Serglycin Heteromer

“…Real-time gelatin zymography was performed as described previously [ 48 , 65 ]. Briefly, zero--point-one percent ( w / v ) MDPF-fluorescent labelled gelatin was incorporated in the 7.5% SDS-PAGE separating gel, and 0.2% ( w / v ) MDPF-fluorescent labelled gelatin was incorporated in the 4.0% SDS-PAGE stacking gel.…”

“…Briefly, zero--point-one percent ( w / v ) MDPF-fluorescent labelled gelatin was incorporated in the 7.5% SDS-PAGE separating gel, and 0.2% ( w / v ) MDPF-fluorescent labelled gelatin was incorporated in the 4.0% SDS-PAGE stacking gel. The fluorescent dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) was used to label gelatin to give MDPF-gelatin as described previously [ 65 ]. The main difference between normal gelatin zymography and real-time gelatin zymography is that in real-time zymography, the gel is not stained, and hence, it is possible to follow the degradation of the gelatin in real time without staining.…”