MMP2/9 enzyme activity was measured by zymography using Novex Zymogram Gelatin Gels (Thermo Fisher Scientific). Supernatant was diluted in Novex Tris-Glycine SDS sample buffer (Thermo Fisher Scientific) and ran on zymogram gel. After electrophoresis, gels were incubated with Novex zymogram renaturing buffer (Thermo Fisher Scientific) and gels incubated in Novex zymogram developing buffer (Thermo Fisher Scientific) for 18 h at 37°C. After incubation, gels were stained with a Colloidal Blue Staining Kit (Thermo Fisher Scientific).
Novex zymogram gelatin gel
Manufactured by Thermo Fisher Scientific
Sourced in Italy
Novex Zymogram Gelatin Gels are laboratory equipment used to detect and analyze the presence and activity of proteolytic enzymes, such as gelatinases, in biological samples. They provide a platform for the separation and visualization of these enzymes through electrophoresis and subsequent staining.
Automatically generated - may contain errors
Lab products found in correlation
Tris glycine sds native sample buffer, by Thermo Fisher Scientific (4 mentions)
Novex zymogram developing buffer, by Thermo Fisher Scientific (3 mentions)
Novex zymogram renaturing buffer, by Thermo Fisher Scientific (3 mentions)
Novex tris glycine sds sample buffer, by Thermo Fisher Scientific (2 mentions)
Colloidal blue staining kit, by Thermo Fisher Scientific (2 mentions)
Coomassie blue r 250, by Bio-Rad (2 mentions)
Developing buffer, by Thermo Fisher Scientific (2 mentions)
Tris glycine sds running buffer, by Thermo Fisher Scientific (2 mentions)
Zymogram renaturing buffer, by Thermo Fisher Scientific (2 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (2 mentions)
Gel logic 100, by Kodak (1 mentions)
Gel logic 100 imaging system, by Kodak (1 mentions)
Zymogram developing buffer, by Thermo Fisher Scientific (1 mentions)
8 protocols using novex zymogram gelatin gel
1
Zymographic Quantification of MMP2/9 Activity
2
Zymographic Analysis of MMP2 and MMP9 Activity
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MMP2 and MMP9 enzyme activity were measured by zymography using Novex Zymogram Gelatin Gels (Thermo Fisher Scientific). Fibroblast supernatant were diluted in Novex Tris-Glycine SDS sample buffer (Thermo Fisher Scientific) and were run on zymogram gels. After electrophoresis, gels were incubated with Novex zymogram renaturing buffer (Thermo Fisher Scientific) and incubated in Novex zymogram developing buffer (Thermo Fisher Scientific) for 18 hours at 37°C. After incubation, gels were stained with a Colloidal Blue Staining Kit (Thermo Fisher Scientific) and imaged.
3
MMP-9 Activity Quantification via Gelatin Zymography
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MMP-9 activity in a medium derived from rhTGF-β1-treated MRPECs was determined by gelatin zymography. Briefly, the medium was mixed with Tris-Glycine SDS Native Sample Buffer (1:1; Invitrogen) and electrophoresed through 10% Novex Zymogram Gelatin Gels (Invitrogen) with Tris-Glycine SDS Running Buffer (Invitrogen) under constant voltage of 125 V for 120 min. The gel was then incubated in Zymogram Renaturing Buffer (Invitrogen) for 30 min at room temperature with gentle agitation and washed with developing buffer (Invitrogen) for 30 min. The gel was further incubated for 24 h in fresh developing buffer at 37°C. After being developed, the gel was stained with 0.5% (w/v) Coomassie Blue R-250 (Bio-Rad) in 50% (v/v) methanol, 10% (v/v) acetic acid for 30 min at room temperature, and destained as described previously [24 (link)]. Gelatinolytic activity of MMP-9 was visualized as a clear band on a blue background. Band intensity was quantified by densitometry using the ImageJ software. Briefly, zymogram gels were scanned using Kodak Gel Logic 100 imaging system and processed into gray scale. Gray scale images were quantified densitometrically by the measurement of the mean intensity of positive band multiplied by its corresponding area. The optical band intensity was then corrected by subtracting the background intensity of equal area.
4
Quantifying Gelatinolytic MMP-9 Activity
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MMP-9 activity in medium derived from TGF-β treated HKGECs was determined by gelatin zymography. Briefly, medium was mixed with Tris-Glycine SDS Native Sample Buffer (1:1; Invitrogen) and electrophoresed through 10 % Novex Zymogram Gelatin Gels (Invitrogen) with Tris-Glycine SDS Running Buffer (Invitrogen) under constant voltage of 125 V for 120 min. After electrophoresis, gels was incubated in Zymogram Renaturing Buffer (Invitrogen) for 30 min at room temperature with gentle agitation and washed with developing buffer (Invitrogen) for 30 min. The gel was further incubated for 24 h in fresh developing buffer at 37 °C. After developing, the gel was stained with 0.5 % (w/v) Coomassie Blue R-250 (Bio-Rad) in 50 % (v/v) methanol, 10 % (v/v) acetic acid for 30 min at room temperature, and destained as described previously [25 (link)]. Gelatinolytic activity of MMP-9 was visualized as a clear band on a blue background. Band intensity was quantified by densitometry using ImageJ software. Briefly, zymogram gels were scanned using Kodak gel logic 100 imaging system and processed into gray scale. Gray scale images were quantified densitometrically by the measurement of the mean intensity of positive band multiplied by its corresponding area. The optical band intensity was then corrected by subtracting background intensity of equal area.
5
Gelatin Zymography for Protease Activity
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Conditioned media collected from MRC5 and NHDF fibroblasts (10–20 μL) were diluted with 4× Tris–Glycine SDS Native Sample Buffer (Invitrogen, Monza, Italy) and loaded onto 10% Novex Zymogram Gelatin Gels (Invitrogen). After electrophoretic separation, gels were developed following the manufacturer’s instructions. After incubation with SimplyBlue Safestain (Invitrogen) buffer, gelatinolytic activity was detected as transparent bands in the otherwise homogeneous blue gel and quantified using ImageJ software.
6
Gelatin Zymography for MMPs
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Aliquots (10-20 mL) of culture media were mixed with Tris-Glycine SDS Native Sample Buffer (Invitrogen), electrophoresed through 10% Novex Zymogram Gelatin Gels (Invitrogen) and developed according to the manufacturer's instructions. The bands containing gelatinolytic activity of MMP2 and MMP9 appeared transparent in the otherwise blue gel. Bands were quantified using ImageJ software.
7
Gelatin Zymography for Protease Activity
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Total protein homogenates (40 ug, n = 6) from WT and CCR7−/− LV subjected to pressure-overload were mixed with 2× non-reducing SDS-PAGE sample buffer and resolved through a 10% Tris-Glycine gel with 0,1% gelatin (Novex Zymogram Gelatin Gel, Invitrogen, San Diego, CA). After electrophoresis the gels were first incubated in 100 mL Novex Zymogram Renaturing Buffer and then in 100 mL Novex Zymogram Developing Buffer (both Invitrogen, San Diego, CA) for 30 min under gentle agitation at room temperature, before incubating overnight (20–24 hours) in 100 mL Novex Zymogram Developing Buffer at 37°C. The gels were subsequently rinsed 3×5 minutes with deionized water under gentle agitation at room temperature, before they were stained in 20 mL SimplyBlue Safestain (Life Technologies AS) for one hour at room temperature under gentle agitation. Finally, the gels were rinsed 2×1 hour in deionized water, scanned with a resolution of 300 dpi and saved as TIFF images. Band intensities were measured using ImageJ (Wayne Rasband, National Institute of Mental Health).
8
Gelatin Zymography for Protease Activity
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Conditioned media from 2 Â 10 5 BMECs, treated or not with TPO at 10 ng/mL for 16 hours, were collected and mixed with SDS buffer under nonreducing conditions, and run on Novex Zymogram gelatin gel (Invitrogen Corp.) at 125 V for 90 minutes. After electrophoresis, the enzyme was renatured by incubating the gel in Zymogram renaturing buffer containing a nonionic detergent. The gel was equilibrated in Zymogram developing buffer, and then stained and destained following manufacturer's instructions (Invitrogen Corp.). Regions of protease activity appeared as clear bands against a dark blue background.
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