Human recombinant MMP12 (R&D Systems) was activated by 4 hours of incubation at 37°C with 1 mmol/L 4-aminophenylmercuric acetate, in 1× Zymogram Development Buffer (Bio-Rad). Subsequently, 200 nM of human recombinant galectin-3 (R&D Systems) was incubated with either nonactivated pro-MMP12 (100 nM), active MMP12 (100 nM), or active MMP12 (100 nM) plus human recombinant TIMP (tissue inhibitor of metalloproteinase)-3 (10 nmol/L) in 1× Zymogram Development Buffer (Bio-Rad). Samples were incubated at 37°C for 60 minutes; the reaction was then stopped by placing samples on ice. Galectin-3 cleavage was then assessed by Western blotting. To evaluate whether MMP inhibition retards galectin-3 cleavage, macrophages were incubated in serum-free media with or without recombinant TIMP3 (10 nM) or the broad-spectrum MMP inhibitor Batimastat, BB94 (20 nM). After 24 hours of incubation at 37°C, conditioned media were collected and the presence of cleaved galectin-3 (fragment of 22 kDa) assessed by Western blotting.
Zymogram development buffer
Manufactured by Bio-Rad
Sourced in United States
Zymogram development buffer is a laboratory reagent used in the analysis of enzymatic activity. It is designed to facilitate the visualization of enzyme bands in zymography, a technique used to detect and quantify the activity of specific enzymes in a sample. The buffer's core function is to provide the necessary conditions for the development and staining of enzyme-containing gels, allowing for the clear identification and analysis of enzyme activity.
Automatically generated - may contain errors
Lab products found in correlation
Zymogram renaturation buffer, by Bio-Rad (8 mentions)
Zymogram sample buffer, by Bio-Rad (4 mentions)
Mini protean tetra cell system, by Bio-Rad (3 mentions)
Gelatin, by Merck Group (3 mentions)
Gel doc xr system, by Bio-Rad (3 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Gelatin, by Bio-Rad (2 mentions)
Polyacrylamide gels, by Carl Roth (2 mentions)
Nonreducing zymogram sample buffer, by Bio-Rad (2 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (2 mentions)
Nanosep, by Pall Corporation (1 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Zymogram renature buffer, by Bio-Rad (1 mentions)
Casein from bovine milk, by Merck Group (1 mentions)
Las 4000 luminescent image analyzer, by Fujifilm (1 mentions)
Quantity 1 d analysis software, by Bio-Rad (1 mentions)
Image lab software version 5, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Centricon, by Pall Corporation (1 mentions)
22 protocols using zymogram development buffer
1
Galectin-3 Cleavage by MMP12
2
Zymographic Analysis of MMP-2 Activity
3
Quantitative Gelatin Zymography Assay
4
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).
5
Quantifying MMP-2 and MMP-9 Activity in RPE-Choroid
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The activities of MMP-2 and MMP-9 in RPE-choroid homogenates were quantified using gelatin zymography. An 8-μg sample of homogenate supernatant total protein was mixed with a non-reducing sample buffer (62.5 mM Tris-HCl, 10% glycerol, 0.1% bromophenol blue; pH = 6.8) and directly electrophoresed in 9% SDS-PAGE containing 0.1% w/v gelatin. After electrophoresis, gels were washed 4 times in a 2.5% v/v Triton X-100 solution to remove excess SDS (20 min each at room temperature), transferred to a solution (Zymogram Development Buffer, Bio-Rad), and incubated for at least 18 h at 37 °C. Protein fixation was achieved by incubating the gels for 15 min with 50% methanol/7% acetic acid and then washing them for a total of 30 min (six 5-min washes) with distilled water. Afterwards, gels were stained for 1 h with GelCode Blue Stain Reagent (Pierce, Rockford, IL, USA), counterstained with distilled water, and analyzed with ImageQuant TL software (GE Healthcare) after densitometric scanning. The MMP activation ratio for MMP-2 was calculated as active MMP intensity/(active MMP + proMMP) intensity. The MMP-9 activity was also determined using intensity measurements. Each zymography assay was repeated at least three times to ensure accuracy.
6
Gelatinase Activity Assay
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Hec50a shSCRM/AXL cells were starved in serum free media for 48 hours and 25,000 cells were plated into a 96 well plate in SF media. Conditioned media was collected 24 hours later. Equal volumes of conditioned media were run under non-reducing conditions on 10% gelatin zymogram gels (BioRad). After electrophoresis, gels were incubated in zymogram renature buffer (BioRad) for 30 min at RT and then incubated overnight in zymogram development buffer (BioRad) at 37°C. Gels were stained for 30 min with Coomassie Brilliant Blue R250 and destained in 40% methanol and 10% glacial acetic acid.
7
Zymographic Analysis of Caseinolytic Activity
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The caseinolytic activity of peritoneal fluids was analyzed by zymography. Casein from Bovine Milk (0.5 mg/mL, Sigma-Aldrich) was added to 11% SDS polyacrylamide gels, and equal amounts of peritoneal fluids (20 μl per well) was run under non-reducing conditions at 4°C. Gels were washed with PBS containing 2.5% Triton X-100, followed by incubation overnight at 37°C in a zymogram development buffer (Bio-Rad). Then, gels were washed, stained with Coomassie blue, and washed again with a de-staining solution. The gels were analyzed using Luminescent Image Analyzer LAS-4000 (Fujifilm Corporation). The caseinolytic activity was visualized as clear bands against a blue background, with detection sometimes obscured by overlapping of non-reactive proteins. Densitometry was performed using TotalLab TL100 (nonlinear dynamics) image analysis software.
8
Gelatin Zymography for MMP2 and MMP9 Detection
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gelatin zymography was used for the detection of MMP2 and MMP9 on a protein level and the corresponding activity. Supernatant fluids were mixed 1:2 with non-reducing Zymogram sample buffer (BioRad, Munich, Germany). Samples were loaded onto 0.75 mm thick, 7.5% polyacrylamide gels (all components Carl Roth, Karlsruhe, Germany) containing 2 mg/ml gelatin (Merck, Darmstadt, Deutschland). Electrophoresis was carried out on the Mini-PROTEAN Tetra Cell System (Bio-Rad). Afterwards, gels were washed twice for 15 min in Zymogram renaturation buffer and incubated over night in Zymogram development buffer at 37°C (both Bio-Rad). Staining with Coomassie solution and subsequent destaining revealed clear bands originating from MMP activity. Pictures of gels were taken and analyzed with the Gel Doc XR+ System and the Quantity 1D Analysis Software (both Bio-Rad).
9
Gelatin Zymography for Measuring MMP Activity
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The gelatinolytic activity of concentrated conditional media samples was assessed after proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 8% polyacrylamide gels containing 10 mg/mL gelatine as a substrate. Samples (25 µg/well total protein) were mixed with Zymogram Sample Buffer (Bio-Rad) and were subjected to SDS-PAGE at 125 V for 95 min. The gels were washed in 1X Zymogram Renaturation Buffer (Bio-Rad) for 30 min at room temperature to remove SDS and to renature the MMPs. Gels were incubated overnight at 37 °C with 1X Zymogram Development Buffer (Bio-Rad) and stained with 0.5% Coomassie blue R-250 in 40% v/v methanol and 10% v/v acetic acid aqueous solution for 30 min at 37 °C. After destaining the gels, the MMPs gelatinase activity was detected using a ChemiDoc MP System and was quantified by densitometry using Image Lab software version 5.2.1 (Bio-Rad).
10
Gelatin Zymography Protocol for Enzyme Analysis
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The gelatin zymography assay was previously described by HW Yeh et al.47 (link). Conditioned medium from each group was collected and concentrated using a centricon (Pall Corporation, 30 K). Samples were mixed with 2X sample buffer (TransLab, South Korea) and separated by 8% SDS-PAGE containing 0.1% gelatin (Sigma Aldrich). After electrophoresis, gels were washed two times in washing buffer (2.5% Triton-X 100 in TBS) and then incubated overnight at 37 °C in 1X zymogram development buffer (Bio-Rad; 161-0766). After incubation, the gels were washed and stained with EZBlueTM Gel Staining Reagent (Sigma Aldrich; G1041) and then washed until white bands appeared on the blue background.
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