Gelatin zymography was performed as previously described (Thirumangalakudi et al. 2007 (link)) to assess the activity of MMP-9 in BV-2 supernatant following treatments. Briefly, samples were run on 10% Gelatin Zymogram Plus Protein Gels (Invitrogen, Carlsbad, CA, USA). Following electrophoresis, gels were incubated at room temperature (RT) with zymogram renaturing buffer (Novex, Carlsbad, CA, USA) for 30 min, and then incubated with zymogram developing buffer (Novex, Carlsbad, CA, USA) for 30 min. Gels were further incubated with fresh developing buffer overnight at 37 °C. Gels were stained with Coomassie Blue R250 for 1 h, and de-stained in deionized water for 1 day. Images scanned on Epson Scanner (Epson, Long Beach, CA, USA) and analyzed using ImageJ.
Zymogram renaturing buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Zymogram Renaturing Buffer is a specialized buffer used in the process of zymography. It is designed to renature proteins within a gel matrix, allowing for the detection and analysis of enzymatic activities. The buffer facilitates the restoration of the native conformation of proteins, enabling the visualization and quantification of specific enzyme activities.
Automatically generated - may contain errors
Lab products found in correlation
Zymogram developing buffer, by Thermo Fisher Scientific (25 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (7 mentions)
Tris glycine sds running buffer, by Thermo Fisher Scientific (6 mentions)
Tris glycine sds sample buffer, by Thermo Fisher Scientific (5 mentions)
Developing buffer, by Thermo Fisher Scientific (3 mentions)
Zymogram gel, by Thermo Fisher Scientific (3 mentions)
Colloidal blue staining kit, by Thermo Fisher Scientific (3 mentions)
Zymogram gelatin gel, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Gelatin, by Avantor (2 mentions)
Novex tris glycine sds sample buffer, by Thermo Fisher Scientific (2 mentions)
Coomassie blue r 250, by Bio-Rad (2 mentions)
Novex zymogram gelatin gel, by Thermo Fisher Scientific (2 mentions)
Tris glycine sds native sample buffer, by Thermo Fisher Scientific (2 mentions)
Gsk690693, by Selleck Chemicals (1 mentions)
Tak 242, by Selleck Chemicals (1 mentions)
As 605240, by Selleck Chemicals (1 mentions)
Gapdh, by Proteintech (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
31 protocols using zymogram renaturing buffer
1
Gelatin Zymography for MMP-9 Activity
2
Zymogram Analysis of Neutrophil Elastase
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NOVEX 4–16 % zymogram blue casein gels (Life Technologies) were used to detect NE enzymatic activity in sputum samples. Mucin samples were homogenized using a sterile Filtropur syringe filter (0.20 μm pore size). Equal volumes of homogenized sputum samples were loaded on a gel and separated using electrophoresis at 125 V for 2 h. The gel was run in Tris/glycine SDS running buffer under nondenaturing conditions. The separated proteins were renatured using a buffer containing a non-ionic detergent (Novex® Zymogram Renaturing Buffer). Gels were washed twice for 15 min in PBS and equilibrated using a developing buffer (Novex® Zymogram Developing Buffer) containing divalent metal cations for 30 min as described in the manufacturer’s protocol. The gel was then incubated at 37 °C for 20 h in fresh developing buffer for enhanced digestion. Enzymatic activity was visualized as a clear band against a dark background of stained casein. ETT mucus from healthy subjects was used as a positive control. The gels were scanned using a Canon ScanLide 50 scanner and activity was measured by quantification of digested area using Image-J densitometry software.
3
Procyanidin Inhibits TLR4-Mediated Signaling
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Procyanidin was purchased from Aladdin Co. Ltd. (Shanghai, China). Secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Toll-like receptor 4 (TLR-4) inhibitor TAK-242, phosphatidylinositol 3-kinase (PI3K) inhibitor AS-605240, and protein kinase B (Akt) inhibitor GSK690693 were purchased from Selleck Chemicals (Shanghai, China). RAGE antibody 553030 was purchased from Calbiochem (Merck, Darmstadt, Germany). Anti-HMGB1 polyclonal antibody 326052233 was purchased from SHINO-TEST Corporation (Sagamihara City, Japan) and GAPDH was purchased from Proteintech, Wuhan, China. Gelatin was purchased from Amresco (Solon, OH, USA). Zymogram renaturing buffer and developing buffer were purchased from Novex (Carlsbad, CA, USA). FBS was purchased from Gibco (Grand Island, NY, USA), and other cell culture media and supplements were purchased from HyClone (Logan, UT, USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
4
Zymography for Protease Activity
5
Gelatin Zymography for MMP-2 and MMP-9
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Gelatin zymograms were used to assess MMP-2 and MMP-9 levels in ischemic brain homogenates and supernatants of cultured endothelia or mixed glia. The supernatant fractions (500 μL) were concentrated via centrifugation at 21,000× g for 30 min at 4 °C using Centrifugal Concentrator VIVASPIN 500 (10 kDa; Sartorius, Göttingen, GE). Concentrated supernatants and 50 μg of each brain homogenate were mixed with non-denaturing sample buffer (93.75 mM Tris-Cl (pH 6.8), 12.5% glycerol and 2.25% SDS) and separated on a 10% SDS polyacrylamide gel containing 0.1% gelatin as substrate. The gel was incubated with zymogram renaturing buffer (Novex, Carlsbad, CA, USA) for 1 h at room temperature followed by zymogram development buffer (Novex, Carlsbad, CA, USA) for 24 h at 37 °C. To visualize protein bands, the gel was stained with the Colloidal blue staining kit (Invitrogen, Carlsbad, CA, USA).
6
Gelatinase Zymography of Pericyte Secretome
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Gelatinase zymography was performed as previously described with modifications [18 (link)]. Briefly, isolated microvessels were homogenized in lysis buffer, and the pericytes conditioning media (1.5 ml) was concentrated to 20–30 μl using microcon spin columns (Millipore, Bedford, MA). The samples were separated on an 8% polyacrylamide SDS gel containing 1 mg/ml gelatin. The gel was then soaked in zymogram renaturing buffer (Novex, Shanghai, China) for 30 min at room temperature and incubated for 16 h at 37 °C in zymogram developing buffer (Novex, Shanghai, China). To visualize the MMPs, the gel was stained with SimplyBlue Safe Stain (Invitrogen, Shanghai, China) for 30 min. The gel was imaged using a CCD camera and analyzed using ImageJ software (NIH, Bethesda, MD, USA).
7
Comprehensive Antibody Procurement for Protein Assays
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Antibody for β-actin was purchased from Sigma (St. Louis, MO). Antibodies for hypoxia inducible factor-1α (HIF-1α), heatshockprotein70 (HSP70), phosphorylated p38 (Tyr182) were purchased from Cell Signaling Technology (Beverly, MA). TF was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA.). Secondary antibodies were purchased from Sigma (St. Louis, MO). L-OHP was purchased from Jiangsu heng rui medicine (Lianyungang, China). Gelatin was purchased from Amresco (Solon, OH, USA). Zymogram renaturing buffer and Zymogram developing buffer were purchased from Novex (Carlsbad, CA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco, and other cell culture media and supplements were purchased from HyClone (Logan, UT, USA). Coomassie brilliant blue G250 was purchased from Beyotime (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma Chemical Co (St. Louis, MO). All other chemicals were purchased from Sigma Chemical Co (St. Louis, MO). Hirudin was purchased from Genscript (Nanjing, China). p38 inhibitor SB 203580 was purchased from MedChemExpress (NJ, USA).
8
Quantifying Arterial MMP-2 and MMP-9
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Ligated left common carotid arteries were collected, gently flushed to remove all blood clots and immediately snap frozen. Tissues were homogenized in RIPA buffer (Sigma Aldrich, R0278) and protein content was determined using the BCA method (Pierce BCA Protein Assay Kit, Thermo Scientific, 23225). Samples were mixed with Laemmli sample buffer without β-mercaptoethanol before loading on a 10% Zymogram Gelatin gel (Life Technologies, EC6175). After electrophoresis in Tris-Glycine SDS Running Buffer (Life Technologies, LC2675), proteins were renaturated in Zymogram Renaturing Buffer (Life Technologies, LC2670) and incubated with Zymogram Developing Buffer (Life Technologies, LC2671) overnight. Gels were stained with Coomassie brilliant blue (Merck, 115444) for 3 h and subsequently destained twice for 1 h. Finally, gels were scanned to visualize the gelatinolytic activity of MMP9 and MMP2 in the left common carotid artery.
9
Measuring Inflammatory Mediators in BALF
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The concentration of MMP-9 in BALF and neutrophil supernatants was measured using an ELISA, according to the manufacturer's directions (R&D Systems, Minneapolis, MN). To assess MMP-9 gelatinolytic activity, BALF was mixed 1:1 (vol:vol) with Tris-Glycine-SDS Sample Buffer (Life Technologies Ltd, Paisley, UK) and loaded onto 10% zymogram (gelatin) gels (Life Technologies Ltd). Samples were subsequently electrophoresed at 125 V for 90 min in Tris-Glycine-SDS Running Buffer (Life Technologies Ltd). Following electrophoresis, gels were incubated in 1 × Zymogram Renaturing Buffer (Life Technologies Ltd) for 30 min at room temperatrue. To visualize the gelatinolytic activity, the gel was incubated in 1 × Zymogram Developing Buffer (Life Technologies Ltd) overnight at 37 °C before staining with SimplyBlue Safestain (Life Technologies Ltd).
The concentrations of LTA4H, NE and MMP-12 in BALF and/or neutrophil supernatants were measured using an ELISA, according to the manufacturer's directions (USCN Life Science, Hubei, PRC).
The concentrations of LTA4H, NE and MMP-12 in BALF and/or neutrophil supernatants were measured using an ELISA, according to the manufacturer's directions (USCN Life Science, Hubei, PRC).
10
Quantification of Lung Inflammation Mediators
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