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Molecular Analysis in Mechanobiology
Published in Jiro Nagatomi, Eno Essien Ebong, Mechanobiology Handbook, 2018
Zymography is a variation of SDS-PAGE that analyzes the activity of matrix degradative enzymes [71,72]. The method is most widely applied using gelatin as a substrate for MMP-2 and MMP-9, but variations have been developed for other MMPs such as collagenases, as well as hyaluronidases. A substrate for the enzyme of interest is incorporated within the resolving gel and SDS-PAGE performed under nonreducing conditions. The gel is washed in a Triton X-100 buffer to remove SDS and then incubated under appropriate conditions for enzymatic activity. The gel is then stained with Coomassie blue (for protease assays) to reveal clear bands at molecular weights corresponding to specific enzymes. Substrate degradation and corresponding enzymatic activity can be measured semi-quantitatively by densitometry. A further variation, reverse zymography, may also be performed to analyze the activity of tissue inhibitors of matrix metalloproteases (TIMPs) in which both a substrate and a protease are included within the gel and TIMP activity detected as stained bands where degradation has been inhibited.
Visualizing biogeochemical heterogeneity in soils and sediments: A review of advanced micro-scale sampling and imaging methods
Published in Critical Reviews in Environmental Science and Technology, 2023
Cai Li, Shiming Ding, Musong Chen, Zhilin Zhong, Qin Sun, Yan Wang
As PO imaging does not interfere with DGT and zymography measurements, it can be combined with DGT and zymography to map the 2D distribution of chemical parameters, labile solutes, and enzyme activities in soils and sediments (Table S6). Recently, three studies have consecutively used PO, DGT (or DGT-PO), and zymography to study the rhizosphere processes (Bilyera et al., 2022; W. Fang et al., 2021; Zhang et al., 2021). They visualized multiple P mobilization and immobilization processes by mapping the O2, pH, phosphatase activity and labile P, Fe, and Mn in the rhizosphere (Figure S2). These high-resolution imaging methods provide a 2D spatial distribution of both chemical information and biological responses. Therefore, based on spatially high-resolution chemical and biological coupling information, the complex biogeochemical processes of solutes in heterogeneous environments can be revealed.
In vitro characterization of cutaneous immunotoxicity of immortalized human keratinocytes (HaCaT) exposed to reactive and disperse textile dyes
Published in Journal of Toxicology and Environmental Health, Part A, 2018
Daniela Morais Leme, Andrea Sehr, Tamara Grummt, Jenifer Pendiuk Gonçalves, Thiago Jacomasso, Sheila Maria Brochado Winnischofer, Francine Bittencourt Potrich, Carolina Camargo de Oliveira, Edvaldo da Silva Trindade, Danielle Palma de Oliveira
Gelatinolytic activities of MMP-2 and -9 on supernatants of HaCaT cultures treated with tested dyes were detected by gelatin-zymography. Supernatant were mixed with nonreducing sample buffer (Tris–HCl 0.3 M pH 6.8, 50% v/v glycerol, 10% w/v Sodium dodecyl sulfate) and electrophoretically separated in 8% polyacrylamide gel containing 1 mg/ml gelatin (Fluka). Pre-stained molecular weight standard ranging from 3.5 to 260 kDa (Invitrogen) was used for location of the bands. Gels were washed with ultrapure water, followed by three washes with triton X-100 2.5% (VETEC) for 10 min each and five additional washes with water to remove any froth. Gels were then incubated with MMP optimum incubation buffer (0.05 M Tris–HCl, 10 mM CaCl2, 1 µM ZnCl2, pH 8) for 72 h at 37°C, washed with water, and stained with Coomassie Brilliant Blue R-250 0.5% (VETEC) in 30% methanol (Merck): 10% glacial acetic acid (Merck) water solution for 30 min (TOTH; FRIDMAN). The gelatinolytic activities of MMP-2 and -9 as a clear band against dark background of stained gelatin were visualized. Gels were scanned (A3 Transparency Unit EPSON scanner I-88 model) and band intensities quantified using ImageJ software.
Extracellular collagenase isolated from Streptomyces antibioticus UFPEDA 3421: purification and biochemical characterization
Published in Preparative Biochemistry & Biotechnology, 2023
Elizianne Pereira Costa, Romero Marcos Pedrosa Brandão-Costa, Wendell Wagner Campos Albuquerque, Thiago Pajeú Nascimento, Amanda Emmanuelle Sales Conniff, Kethylen Barbara Barbosa Cardoso, Anna Gabrielly Duarte Neves, Juanize Matias da Silva Batista, Ana Lúcia Figueiredo Porto
Gelatin zymography was performed according to Laemmli[38] with some modifications. Separating gel (10%) was prepared containing 1 mg/mL of gelatin and 20% SDS. The purified enzyme samples were applied to the polyacrylamide gel and the electrophoresis was carried out at 4 °C. The gel was then washed in a Triton X-100 (2.5% v/v) solution for 30 min and was incubated overnight in 0.05 M Tris-HCl buffer (pH 7.8) containing 1.0 mM CaCl2 at 37 °C. The gel was stained by silver nitrate.[38]