Detection of proteases in polyacrylamide gels containing covalently bound substrates

Detection of extracellular proteases from microorganisms on agar plates

“…These methods as with other protease assays are based on the hydrolysis of substrate for detection of gelatinaselike proteases and have been used in several methods for biochemical studies (Branquinha et al 1996) and in clinical laboratory for identification of bacteria (MacFaddin 1980). BSA, a smaller molecule, is also frequently used incorporated in SDS-PAGE (Kelleher & Juliano 1984) or in radial diffusion in agar gel (Wikström et al 1981). Denatured hemoglobin is routinely used in assay systems for detecting proteolytic activity (Anson 1939) and also incorporated in SDS-PAGE (Andary & Dabich 1974).…”

The Detection of Enzyme Activity Following Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

“…By polymerizing the running gel in the presence of substrate molecules, it is often possible to immobilize macromolecules within the polyacrylamide matrix in a form accessible and recognizable to enzymes. Such immobilization can result from either the formation of direct, covalent linkages between the macromolecule and the polyacrylamide matrix itself or through physical entrapment within the gel matrix (132). This approach provides a means of overcoming the slow rate of diffusion of many proteins, oligosaccharides, and oligonucleotides into the polymerized gel and ensures that the product formed remains localized.…”

Proteolytic activity in infected and noninfected insect cells: Degradation of HIV-1 Pr55gag particles

“…The complex specificity of proteases is clear from the diversity of the substrates and methods that have been developed for their assays: spectrophotometry (Feder, 1968), fluorescence (Auld and Prescott, 1983;Bratovanova and Petkov, 1987;Kuromizu et al, 1985;Tisljar and Denker, 1986), HPLC (Green, 1986;Kuwada and Katayama, 1984;Nagatsu et al, 1985), ELISA (Ohlsson et al, 1986;Yoshioka et al, 1987), and methods using gel-entrapped substrates (Brown et al, 1982;Kelleher and Juliano, 1984;Nagase and Woessner, 1980) or luminogenic and chromogenic substrates (Branchini et al, 1980). When there is no information about the proteases involved, the choice of the method and substrate becomes an even larger problem.…”