Zymography was performed as previously described [28 (link)]. Briefly, protein samples were equally loaded and separated by a 10 % Tris-glycine gel with 0.1 % gelatin as a substrate. After separation, the gel was washed in distilled water twice for 30 min, re-natured for 1 h with 2.5 % Triton X-100 buffer at room temperature, and incubated for 48 h with developing buffer (0.05 M Tris-HCl, pH 7.5; 0.2 mol/L NaCl; 5 mmol/L CaCl2; 0.05 % Brij-35; and 0.2 mmol/L NaN3) at 37 °C. Following this, the gel was stained with 0.05 % Coomassie R-250 dye (Sigma) for 30 min and appropriately de-stained. Gelatinolytic activity (MMP-9, 92 kDa) was determined as by the appearance of clear bands at the appropriate molecular weights.
Coomassie r 250 dye
Manufactured by Merck Group
Sourced in United States
Coomassie R-250 dye is a synthetic dye used in biochemical applications. It is a popular staining agent for detecting and visualizing proteins in polyacrylamide gel electrophoresis (PAGE) and other analytical techniques.
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Lab products found in correlation
6 protocols using coomassie r 250 dye
1
Zymographic Analysis of Gelatinase Activity
2
Gelatin Zymography for Gelatinase Activity
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Equal amounts of protein from the cerebral vessel samples were loaded and separated on a 10% Tris-glycine gel with 0.1% gelatin as the substrate. Then, the gel was washed and renatured with 2.5% Triton X-100 buffer. After incubation with developing buffer at 37 °C for 24 h, the gel was stained with 0.05% Coomassie R-250 dye (Sigma-Aldrich, MO, USA) for 30 min and de-stained. The gelatinase standard was mixed with pro-MMP-9 and pro-MMP-2 (Sino Biological Inc., Beijing, China)70 (link). Pro-MMP-9 activity was evaluated by optical density.
3
Gelatin Zymography for MMP Detection
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Zymography was performed as previously described [25 (link)]. Briefly, equal amounts of protein were loaded and separated on a 10% Tris-glycine gel with 0.1% gelatin as the substrate. Then, the gel was washed and renatured with 2.5% Triton X-100 buffer. After incubation with developing buffer at 37 °C for 24 h, the gel was stained with 0.05% Coomassie R-250 dye (Sigma-Aldrich) for 30 min and destained. Gelatinolytic activity (MMP-9: ~ 97 kDa; MMP-2: ~ 72 kDa) was determined as clear bands at the appropriate molecular weights.
4
Zymography for MMP2 and MMP9 Assessment
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Zymography was performed as previously described 22 (link). Briefly, equal amounts of protein from abdominal aortic samples were loaded and separated on a 10% Tris-glycine gel with 0.1% gelatin as the substrate. Then, the gel was washed and renatured with 2.5% Triton X-100 buffer. After incubation with developing buffer at 37 °C for 24 h, the gel was stained with 0.05% Coomassie R-250 dye (Sigma-Aldrich, MO, USA) for 30 min and destained. MMP2 and MMP9 activity levels were evaluated by measuring the optical density.
5
Gelatin Zymography for MMP-9 Detection
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Zymography was performed as previously described [72 (link)]. Briefly, protein samples were prepared similar to Western blot but without boiling. Samples were equally loaded and separated by 10% triglycine gel with 0.1% gelatin as the substrate. After separation, the gel was washed in distilled water, re-natured for 1 h with 2.5% Triton X-100 buffer at room temperature, and incubated for 48 h with developing buffer (0.05 M Tris-HCl pH 7.5, 0.2 mol/L NaCl, 5 mmol/L CaCl2, 0.05% Brij-35, 0.2 mmol/L NaN3) at 37°C. Then, the gel was stained with 0.05% Coomassie R-250 dye (Sigma-Aldrich) for 30 min and appropriately de-stained. Gelatinolytic activity (MMP-9, 97 kDa) was determined as showing clear bands at the appropriate molecular weights.
6
Gelatin Zymography for MMP-9 Activity
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Zymography was performed as previously described [23] (link). Briefly, equal amounts of protein were separated on a 10% Tris-glycine gel copolymerized with 0.1% gelatin as substrate. After separation, the gel was washed twice in distilled water (30 min each wash) and then, proteins within the gel were renatured by incubation with 2.5% Triton-X-100 buffer for 1 h at room temperature. After incubating with developing buffer (0.05 M Tris-HCl pH 7.5, 0.2 M NaCl, 5 mM CaCl2, 0.05% Brij-35, and 0.2 mM NaN3) at 37°C for 24 h, the gel was stained with 0.05% Coomassie R-250 dye (Sigma) for 30 min followed by destaining. The recombinant mouse MMP -9 (Abcam plc, Cambridge, UK) was used as a positive control to identify the active form of MMP-9. Gelatinolytic activity (MMP-9: ∼89 kDa) was determined as clear bands at the appropriate molecular weight.
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