Nomilin (purity > 98%) was purchased from Shanghai aladdin Medical Technology Co., Ltd. Recombinant human IL‐1β, dimethylsulphoxide (DMSO) and type II collagenase were purchased from Sigma‐Aldrich. The primary antibody against collagen Ⅱ, Lamin B1, iNOS, COX‐2 and GAPDH was acquired from Abcam, goat anti‐rabbit and antimouse IgG‐HRP were from Bioworld and antibodies against Keap1, Nrf2, HO‐1, COX‐2, IκBα and p65 were purchased from Cell Signaling Technology; Alexa Fluor®488 labelled and Alexa Fluor®594 labelled Goat Anti‐Rabbit IgG (H + L) second antibody was purchased from Jackson ImmunoResearch. The 4′, 6‐diamidino‐2‐phenylindole (DAPI) was obtained from Beyotime. The cell culture reagents were purchased from Gibco. Foetal bovine serum (FBS), bovine serum albumin (BSA), Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium and 0.25% trypsin‐ethylenediaminetetraacetic acid (trypsin–EDTA) were purchased from Gibco (Life Technologies Corp.). TRIzol reagent was purchased from Invitrogen. Quanti Tect Reverse Transcription kit was purchased from Qiagen. SYBR Green Master Mix was purchased from Bio‐Rad Laboratories. ELISA kits of PGE2, TNF‐α, IL‐6, Collagen II, Aggrecan, MMP‐13 and ADAMTS‐5 were purchased from R&D systems. Griess reagent was purchased from Beyotime Institute of Biotechnology.
Adamts 5
Manufactured by R&D Systems
Sourced in United States
ADAMTS-5 is a member of the ADAMTS (A Disintegrin And Metalloproteinase with Thrombospondin Motifs) family of enzymes. It is a metalloproteinase that cleaves the major cartilage proteoglycan, aggrecan. The core function of ADAMTS-5 is the breakdown of the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Adamts 4, by R&D Systems (4 mentions)
Elisa kits of pge2, by R&D Systems (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Sybr green master mix, by Bio-Rad (2 mentions)
Griess reagent, by Beyotime (2 mentions)
Mmp 13, by R&D Systems (2 mentions)
Collagenase type 2, by Merck Group (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Aggrecan, by R&D Systems (1 mentions)
Goat anti rabbit and antimouse igg hrp, by Bioworld Technology (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Monosodium iodoacetate, by Merck Group (1 mentions)
Timp 3, by R&D Systems (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Mmp 3, by R&D Systems (1 mentions)
Carboxymethylcellulose, by Merck Group (1 mentions)
Recombinant human il 1β, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
8 protocols using adamts 5
1
Osteoarthritis Mechanism Investigation
2
Naringenin-based Osteoarthritis Treatment
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Naringenin (C15H12O5) (4′,5,7-trihydroxyflavanone, molecular weight of 272.26, 95%purity, CAS number: 67604-48-2) was purchased from Glentham Life Sciences Ltd., Edinburgh, UK. The Amphorae coffeaeformis powder was supplied by members of Algal Biotechnology Unit (National Research Centre, Dokki, Giza, Egypt). Malondialdehyde (MDA) and reduced glutathione (GSH) assaying commercial diagnostic kits were purchased from the Biodiagnostic Company for Research Kits in Egypt. ELISA kits for rat disintegrin and metalloproteinase with thrombospondin 5 repeats (ADAM TS-5) (catalog number: SEK205Ra), matrix metalloproteinase-3 (MMP-3) (catalog number: LS-F5516.) and rat tissue inhibitor of metalloproteinase-3 (TIMP-3) (catalog number: RK03988) were provided by R&D System, Minneapolis, MN, USA. Indomethacin, monosodium iodoacetate, and all other compounds with high analytical grades were supplied by Sigma Chemical Company (St. Louis, MO, USA).
3
Sinapic Acid Anti-Inflammatory Effects
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Sinapic acid (purity ≥98%), carboxymethylcellulose, collagenase type II, recombinant human IL‐1β and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). SA was dissolved in 0.5% carboxymethylcellulose sodium to form oral suspension. Cell‐counting kit‐8 (CCK‐8) was obtained from Dojindo (Kumamo, Japan). Primary antibodies against COX‐2 and iNOS were purchased from Abcam (Cambridge, MA). Primary antibodies against p65, inhibitor of kappa B (IκB)‐α, Nrf2 and HO‐1 were acquired from CST (Danvers, MA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA). Griess reagent was acquired from Beyotime Institute of Biotechnology (Shanghai, China). SYBR Green Master Mix was purchased from Bio‐Rad Laboratories (Hercules, CA). QuantiTect Reverse Transcription kit was purchased from Qiagen (Valencia, CA). ELISA kits of PGE2, TNF‐α, IL‐6, MMP‐13 and ADAMTS‐5 were purchased from R&D systems (Minneapolis, MN).
4
Quantitative Protein Analysis of Cartilage
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Cellular protein from cells or tissue prepared using buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) was separated by SDS-PAGE following standard protocols (Amersham BioSciences, Piscataway, NJ, USA). The antibodies used for Western blotting were as follows: aggrecan (ab3778; Abcam, Cambridge, UK), collagen type II (ab3778; Abcam), MMP-13 (sc-515,284; Santa Cruz Biotechnology, Dallas, TX, USA), ADAMTS-4 (MAB4307; R&D Systems, Minneapolis, MN, USA), ADAMTS-5 (MAB2198; R&D Systems), and GAPDH (sc-166,574; Santa Cruz). An enhanced chemiluminescence system (Amersham BioSciences) was used for detection. Blots were quantified using the ImageJ densitometry program (National Institutes of Health, Bethesda, MD, USA).
5
Fluorometric Enzyme Activity Assay
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Fluorescence and
UV–vis spectra were collected with a HORIBA FluoroMax-4 spectrofluorometer
and Cary 60 UV–vis spectrophotometer, respectively. Pyropheophorbide-a acid (pyro) was prepared according to literature.42 (link) Black hole quencher-3 carboxylic acid succinimidyl
ester (BHQ3-NHS) was purchased from LGC Biosearch Technologies. Recombinant
human MMP-13, ADAMTS-4, and ADAMTS-5 enzymes were purchased from R&D
Systems (Minneapolis, MN). MMP Multipacks were purchased from Enzo
Life Sciences. Proteinase K and singlet oxygen sensor green (SOSG)
were purchased from Thermo Scientific and Invitrogen, respectively.
Peptides on solid support resin were purchased from GL Biochem (Shanghai)
Ltd. MMP buffer comprised 50 mM HEPES, 10 mM CaCl2, and
0.05% Brij 35, pH 7.5. ADAMTS buffer contained 50 mM Tris, 100 mM
NaCl, 5 mM CaC2, and 0.05% Brij 35, pH 7.5. All other reagents
were purchased from Sigma (St. Louis, MO).
UV–vis spectra were collected with a HORIBA FluoroMax-4 spectrofluorometer
and Cary 60 UV–vis spectrophotometer, respectively. Pyropheophorbide-a acid (pyro) was prepared according to literature.42 (link) Black hole quencher-3 carboxylic acid succinimidyl
ester (BHQ3-NHS) was purchased from LGC Biosearch Technologies. Recombinant
human MMP-13, ADAMTS-4, and ADAMTS-5 enzymes were purchased from R&D
Systems (Minneapolis, MN). MMP Multipacks were purchased from Enzo
Life Sciences. Proteinase K and singlet oxygen sensor green (SOSG)
were purchased from Thermo Scientific and Invitrogen, respectively.
Peptides on solid support resin were purchased from GL Biochem (Shanghai)
Ltd. MMP buffer comprised 50 mM HEPES, 10 mM CaCl2, and
0.05% Brij 35, pH 7.5. ADAMTS buffer contained 50 mM Tris, 100 mM
NaCl, 5 mM CaC2, and 0.05% Brij 35, pH 7.5. All other reagents
were purchased from Sigma (St. Louis, MO).
6
Fibulin-2 Proteolytic Assay with ADAMTS and MMP Enzymes
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Proteolytic assay on Fibulin-2 (0.5 μg per reaction, kindly provided by Dr. T. Sasaki, Oita University, Japan; or purchased from Abnova, ref. 28–1184), was carried out in 50 μL of reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij 35, pH 8.5). Recombinant ADAMTS-1, ADAMTS-4, ADAMTS-5 and MMP-2 were purchased from R&D Systems and employed at 50 nM. Reactions were allowed to proceed at 37°C for the indicated times and stopped by addition of reducing SDS-sample buffer containing 20 mM EDTA. Then, digestion products were visualized by Western blot as described above. When indicated, samples were preincubated with ADAMTS-12 for 30 min before addition of ADAMTS-5. Production and purification of recombinant ADAMTS-12 was performed as previously described [56 (link)].
7
Quantification of MMP and ADAMTS Levels
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The expressions of pro-form MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5 were determined using ELISA assay. MMP-1 and MMP-13 kits were purchased from Elabscience, China. ADAMTS-4 and ADAMTS-5 were purchased from R&D Systems, USA. In brief, 20, 000 T/C-28a2 cells were plated on a 96-well plate. After necessary treatment, the supernatant was collected from the treated cells. The supernatant was added into the 96-well plate together with the standards to be incubated for 1 hour, followed by removing the medium and adding the conjugate solutions. After 30 minutes of incubation, the TMB solution was introduced to be incubated for 15 minutes, followed by adding the stop solution. Lastly, a microplate reader was used to measure the absorbance at 450 nm.
8
Neurocan Degradation by ADAMTS
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Neurocan degradation (0.5 μg) was performed using recombinant mouse neurocan protein (R&D Systems, Ref. 5800-NC) in 30 μL reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl 2 , 0.05% Brij 35, pH 8.5). Recombinant ADAMTS1 (Ref. 2197-AD), ADAMTS4 (Ref. 4307-AD), or ADAMTS5 (Ref. 2198-AD), purchased from R&D Systems, was added at 50 nM. Recombinant ADAMTS12 was obtained as described previously [19] . The reactions were allowed to proceed at 37 °C for the indicated times, 1 mU chondroitinase ABC (Sigma-Aldrich) was added for 30 min, and the reactions were stopped by the addition of a reducing SDS-sample buffer containing 20 mM EDTA. Digestion products were visualized by western blot analysis.
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