The proteinase activities of MMP-9 and MMP-2 were measured by gelatin zymography. 10% polyacrylamide gels copolymerized with gelatin (0.1%, G8150, Sigma) were prepared. H9c2 (5 × 104 cells/mL) and HepG-2 (5 × 105 cells/mL) cells were seeded at volumes of 0.5 mL in 24 well plates. After being pretreated with PNG extracts/fractions for 24 h, the cells were stimulated by recombinant rat TNF-α (10 ng/mL) for another 72 h. The supernatants were collected and loaded into each well of the same volume. Following 2 h of electrophoresis, the gels were washed with 2.5% Triton X-100 for 1 h at room temperature to remove sodium dodecyl sulfate (SDS). Gels were then incubated overnight at 37 °C in renaturing buffer (50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl2, pH 7.5). After incubation, the gels were stained with 0.05% Coomassie Brilliant Blue (G-250, Sigma) in a mixture of methanol: acetic acid: water (5:1:4, v/v) and destained in the same mixture without Coomassie Brilliant Blue. Gelatinolytic activities were detected as transparent bands against the dark blue background. Zymograms were digitally scanned, and band intensities were quantified using GelQuantNET software V 1.7.8.
G 8150
Manufactured by Merck Group
Sourced in United States
The G-8150 is a laboratory equipment product manufactured by Merck Group. It is a multi-purpose device designed for use in various scientific and research applications. The core function of the G-8150 is to provide precise and reliable measurements, but a detailed description while maintaining an unbiased and factual approach is not available.
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3 protocols using g 8150
1
Measuring Proteinase Activities of MMP-9 and MMP-2
2
Gelatin Zymography for Detecting Gelatinases
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Gelatin zymography was used to detect gelatinases in the supernatants of treated cells as has been done by others [19 (link)]. Briefly, supernatants of treated cells (a total of 25 μl per well containing v/v 1:1 of supernatant and Laemmli buffer) were loaded on a gelatin (type A, G-8150, Sigma-Aldrich) containing SDS polyacrylamide gels (acrylamide concentration of 8%) followed by electrophoresis for 6 h. SDS and Triton X-100 were then washed out of the gels to allow a renaturation of the MMP proteins. This was followed by incubation for 24 h (if not specified as prolonged incubation of 36 h) in a zinc- and calcium-chloride-containing buffer at 37 °C, allowing gelatin degradation by gelatinases. Gels were stained with Coomassie blue for 2 h and then destained for 45 to 90 min until bands representing gelatin degradation by the gelatinases became visible. Reconstituted lyophilized human pro-MMP-2 and pro-MMP-9 were used as positive controls (contained in Biotrak MMP-2 and MMP-9 activity assays kits, GE Healthcare). The gelatin zymograms were quantified with the Image-J software (imagej.nih.gov/ij ).
3
Gelatinolytic Zymography for MMP-2 and MMP-9
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MMP-2 and MMP-9 activities were measured in ventricle and aorta by gelatinolytic zymography. Heart or aorta tissues were homogenized in 50 mM Tris buffer, pH 7.4, containing 5 mM CaCl 2 , 1 µM ZnCl 2 and 1% (v/v) Triton X-100. 50 µg of protein were applied to a non-reduced sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis copolymerized with 0.1% (w/v) gelatin (G-8150, Sigma Aldrich, St.
Louis, MO, USA), substrate for MMP-2 and MMP-9, and the zimography was carried out as previously described [18] . Gels were run in a Mini Protean-3 (Bio-Rad Laboratories, Hercules, CA, USA) and incubated for 18 h in 0.15 M NaCl, 10 mM CaCl 2 , 50 Mm Tris HCl pH: 7.4 at 37°C. After staining with Coomassie blue R-250 (B-0149, Sigma Aldrich, St. Louis, MO, USA) and distained with acetic acid-methanolwater (1:3:6), enzyme activity was demonstrated by the absence of staining in areas where the gelatin had been degraded. Pro MMP-2 (72 kDa) and MMP-9 (84 kDa) were identified by molecular weight. Conditioned media from the promyelocyte U-937 cell lines was used as activity standard. The CV intra-assay was < 4.8%, and CV inter-assay < 8.6%. Because of the complexity of this assay, the CV is considered as quite satisfactory. Band intensities were quantified using Sion-Image J software (Scion
Louis, MO, USA), substrate for MMP-2 and MMP-9, and the zimography was carried out as previously described [18] . Gels were run in a Mini Protean-3 (Bio-Rad Laboratories, Hercules, CA, USA) and incubated for 18 h in 0.15 M NaCl, 10 mM CaCl 2 , 50 Mm Tris HCl pH: 7.4 at 37°C. After staining with Coomassie blue R-250 (B-0149, Sigma Aldrich, St. Louis, MO, USA) and distained with acetic acid-methanolwater (1:3:6), enzyme activity was demonstrated by the absence of staining in areas where the gelatin had been degraded. Pro MMP-2 (72 kDa) and MMP-9 (84 kDa) were identified by molecular weight. Conditioned media from the promyelocyte U-937 cell lines was used as activity standard. The CV intra-assay was < 4.8%, and CV inter-assay < 8.6%. Because of the complexity of this assay, the CV is considered as quite satisfactory. Band intensities were quantified using Sion-Image J software (Scion
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