MMP activities in the plasma were assessed using a gelatin zymography assay described previously (Eshaq and Harris, 2019 (link)). Briefly, equal amounts of plasma samples prepared in Zymogram Sample Buffer (Bio-Rad) were separated by SDS-PAGE with 1% gelatin-containing gel. The gel was incubated with Zymogram Renaturation Buffer (Bio-Rad) for 2 h at room temperature and then further incubated in Zymogram Development Buffer (Bio-Rad) overnight at 37°C. The gel was stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich). After destaining, the digested substrate area in the gel was captured and quantified using Image acquisition system and ImageJ, respectively.
Zymogram sample buffer
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Zymogram sample buffer is a solution designed for use in zymography techniques, which are used to detect and analyze enzymatic activities in biological samples. The buffer is formulated to prepare samples for electrophoresis, preserving the enzymatic activity of the proteins present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Renaturation buffer, by Bio-Rad (6 mentions)
Zymogram renaturation buffer, by Bio-Rad (5 mentions)
Zymogram development buffer, by Bio-Rad (4 mentions)
Gelatin, by Bio-Rad (4 mentions)
Development buffer, by Bio-Rad (3 mentions)
Coomassie brilliant blue r 250 staining solution, by Bio-Rad (2 mentions)
Precast gel, by Thermo Fisher Scientific (2 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (2 mentions)
Gelatin, by Merck Group (2 mentions)
Zy00100box, by Thermo Fisher Scientific (2 mentions)
V700 photo scanner, by Epson (2 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Gelatin, by Thermo Fisher Scientific (1 mentions)
Coolpix camera, by Nikon (1 mentions)
Coomassie blue r 250, by Bio-Rad (1 mentions)
Zymography development buffer, by Bio-Rad (1 mentions)
Recombinant mouse mmp 9, by AnaSpec (1 mentions)
Abc peroxidase system, by Vector Laboratories (1 mentions)
28 protocols using zymogram sample buffer
1
Quantitative Gelatin Zymography Assay
2
Zymography of Macrophage-secreted Proteases
3
Gelatin Zymography Analysis of Secreted Proteases
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Cultured media were concentrated 50‐fold using Amicon Ultracel‐ 10K centrifugal filter (Millipore, UFC801096). Concentrated media samples were mixed with zymogram sample buffer (BioRad, 161‐0764). The proteins were separated in 7.5% polyacrylamide gels containing SDS and 1 mg/mL gelatin (BioRad, 170‐6537). After electrophoresis gels were washed (2 × 30 minutes) with zymogram renaturation buffer (BioRad, 161‐0765) while on gentle rotation. Gels were further kept in zymogram development buffer (BioRad, 161‐0766) for 30 minutes at room temperature with gentle shaking. Thereafter, gels were kept in development buffer at 37°C with gentle shaking overnight. Next day, gels were stained with Coomassie blue stain, destained until bands started to appear and images were captured using gel documentation system (ChemiDoc XRS+, BioRad).
4
Zymography Assay for Gelatinase Activity
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Sterile-filtered media were diluted 1:1 with zymogram sample buffer (BioRad) and 20 μl loaded into each well of a Tris-Glycine Novex 10% zymogram protein gel copolymerized with 0.1% gelatin (Invitrogen). SDS-Page gels were run at 118V constant voltage for 90 minutes. gelatinase activity was detected following the manufacturer’s protocol. Images of the gel demonstrating cleared regions of enzymatic activity were captured using a light box and Nikon CoolPix camera. Digital images were quantitated using ImageJ software [28 (link)]. NHDFs were cultured in CC-FGM with or without 50 μM of the MMP inhibitor GM6001 (http://www.emdmillipore.com ) for 72 hours.
5
Articular Cartilage Biomarker Quantification
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Articular cartilage was harvested 21 days after the MIA injection was administered. Each joint cartilage tissue was homogenized in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.1% deoxycholic acid, 2 μg/mL leupeptin, 2 μg/mL aprotinin, and 1 mM PMSF; pH. 7.4) for 3 h at 4°C. The homogenate was centrifuged at 15,000 ×g for 15 min at 4°C. The supernatant was removed and 20–30 μg of it was diluted in Zymogram Sample Buffer (161–0764; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The diluted samples were stored at −70°C until analysis. The levels of gelatinase A (MMP-2), gelatinase B (MMP-9), and COX-2 in the samples were measured using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.
6
Gelatin Zymography for MMP-9 Detection
7
Gelatin Zymography for MMP-9 Detection
8
Zymography for Extracellular Proteases
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Cells were cultured in the absence of serum for 24 hours. The culture medium was then collected and centrifuged for 2 min at 6000 rpm, then combined with zymogram sample buffer (BioRad, Hercules, CA), and incubated at room temperature for 10 minutes. Samples were resolved on a 7.5% SDS-PAGE gel containing 1 mg/ml gelatin. The gel was then incubated in 2.5% Triton X-100 at room temperature for 40 minutes with shaking, and rinsed with incubation buffer (50 mM Tris-B pH8.0; 150 mM NaCl; 10 mM CaCl; 0.05% NaN3), then soaked in incubation buffer at 37°C for 20~24 hours in a shaking waterbath. The gel was then rinsed three times with dH2O, stained with Coomassie blue for 40 minutes at room temperature, and destained at room temperature for 2 hours.
9
Gelatin Zymography for MMP Activity
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To measure MMP activity, we used gelatin zymography. Forty-eight hours after transfection of miRNAs, cells were washed with phosphate-buffered saline (PBS) and then cultured in 300 µL serum-free medium for 24 h. Subsequently, cell-conditioned media was collected and centrifuged at 12,000× g for 10 min at 4℃. The resulting supernatant was mixed with 3 µL zymogram sample buffer (Bio-Rad, CA, USA), and 6 µL of the mixture was loaded onto 8% acrylamide:bisacrylamide (29:1) (Bio-Rad, CA, USA) separating gels containing 0.625 mg/mL gelatin. After electrophoresis at 95 V for 2.5 h, the gels were incubated in renaturation buffer (2.5% Triton X-100) for 2 h at room temperature to remove sodium dodecyl sulfate (SDS). Subsequently, the zymo-gels were washed three times with water, incubated at 37℃ overnight in development buffer (50 mM Tris HCl, pH 7.5, 0.2 M NaCl, 5 mM CaCl, 0.02% Brij 35). After incubation, zymogram gels were stained with 0.25% (w/v) Coomassie brilliant blue R-250 (Bio-Rad, CA, USA) and then destained with destaining buffer (10% acetic acid and 20% methanol).
10
Zymography and Western Blot Analysis of MMP-9
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