MMP and cathepsin gene expression was performed by quantitative PCR (qPCR) using validated Taqman probes (Life Technologies/Applied Biosystems, Carlsbad, CA). All qPCR results are represented as relative quantification (RQ) compared to the mock treated animals and corrected to actin levels. Several MMP levels were measured in BALF and cell culture supernatants using a bead assay (MILLIPLEX MAP MMP Magnetic Bead Panels, EMD Millipore, Billerica, MA) with the BioRad Bio-Plex 200 system (BioRad, Hercules, CA). Cathepsin tissue levels were determined by immunoblots and BALF cathepsin S and B activity assays as previously described46 (link). Cathepsin G BALF activity was determined by a commercial available colorimetric assay (Abcam; ab126780). BALF collagenase activity was determined by a colorimetric ninhydrin method, as previously described22 (link). Proteinase activity was also assessed with the same colorimetric ninhydrin method but utilizing casein as the substrate. BALF elastase activity was measured by the hydrolysis of N-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide(Suc Ala3NA) per minute at 25°C and pH 8.0. BALF gelatinase activity was determined by gelatin zymography.
Ab126780
Manufactured by Abcam
Ab126780 is a laboratory equipment product. It is a device designed for use in scientific research and analysis. The core function of this product is to facilitate specific experimental or measurement tasks within a laboratory setting.
Automatically generated - may contain errors
4 protocols using ab126780
1
Comprehensive Enzymatic Activity Profiling
2
Comprehensive Enzymatic Activity Profiling
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MMP and cathepsin gene expression was performed by quantitative PCR (qPCR) using validated Taqman probes (Life Technologies/Applied Biosystems, Carlsbad, CA). All qPCR results are represented as relative quantification (RQ) compared to the mock treated animals and corrected to actin levels. Several MMP levels were measured in BALF and cell culture supernatants using a bead assay (MILLIPLEX MAP MMP Magnetic Bead Panels, EMD Millipore, Billerica, MA) with the BioRad Bio-Plex 200 system (BioRad, Hercules, CA). Cathepsin tissue levels were determined by immunoblots and BALF cathepsin S and B activity assays as previously described46 (link). Cathepsin G BALF activity was determined by a commercial available colorimetric assay (Abcam; ab126780). BALF collagenase activity was determined by a colorimetric ninhydrin method, as previously described22 (link). Proteinase activity was also assessed with the same colorimetric ninhydrin method but utilizing casein as the substrate. BALF elastase activity was measured by the hydrolysis of N-succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide(Suc Ala3NA) per minute at 25°C and pH 8.0. BALF gelatinase activity was determined by gelatin zymography.
3
Quantifying Neutrophil Elastase in Biological Samples
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The NE footprint Aα-Val360 was measured in plasma samples using a highly specific assay as described previously (28 (link)). BALF NE activity was determined using 50 μM fluorogenic substrate N-(methoxysuccinyl)-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (Enzo Life Sciences) in 0.1 M N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid, 0.5 M NaCl, pH 7.5 by excitation at 360 nm, and emission at 460 nm. Experiments were performed with and without NE inhibitor (1 mM N-[methoxysuccinyl]-Ala-Ala-Pro-Val-chloromethyl ketone). NE was also confirmed by performing immunoblots on BALF using an anti-NE polyclonal antibody (ab68672; Abcam). Cathepsin G activity was determined in 50 μl BALF with a colorimetric cathepsin G activity assay kit (ab126780; Abcam), according to the manufacturer’s instructions.
4
Cathepsin Enzyme Activity Assays
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Enzyme activity assays for cathepsin B (cat# ab65300, Abcam), cathepsin D (cat# ab65302, Abcam), and cathepsin G (cat# ab126780, Abcam) were performed on three snap-frozen MDOTSCC samples of the cohort of six patients used for NanoString mRNA analysis, according to the manufacturer’s protocol. A snap-frozen tonsil sample was used as an appropriate positive control for the cathepsins B (17 (link)) and D (18 (link)) assays, and a denatured sample of the same tonsil was used as an appropriate negative control. The capthepsin G assay kit was supplied with its positive and negative controls. The results were obtained using the Variskan Flash plate reader (Thermo Fisher Scientific). Experiments were performed in duplicates with averages taken for each.
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