Adam10

Manufactured by R&D Systems

Sourced in United States

ADAM10 is a laboratory protein that functions as a metalloprotease enzyme. Its primary activity is the cleavage of membrane-bound proteins, facilitating the shedding of their extracellular domains. ADAM10 plays a role in various physiological processes, including cellular signaling and development.

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Lab products found in correlation

Adam17, by R&D Systems (4 mentions) Mmp 13, by R&D Systems (4 mentions) Actinonin, by Merck Group (3 mentions) Mmp 2, by R&D Systems (3 mentions) Mmp 8, by R&D Systems (3 mentions) Mmp 10, by R&D Systems (3 mentions) Mmp 14, by R&D Systems (3 mentions) 930 adb, by R&D Systems (3 mentions) Mmp 9, by R&D Systems (3 mentions) Mmp 1, by R&D Systems (3 mentions) Es010, by R&D Systems (2 mentions) Nff449, by Bio-Techne (2 mentions) Cxcl10, by Thermo Fisher Scientific (2 mentions) Mmp 2, by Merck Group (2 mentions) Mmp 13, by Merck Group (2 mentions) Mmp 9, by Merck Group (2 mentions) Mmp 8, by Merck Group (2 mentions) Tcrβ h57 597, by BioLegend (1 mentions) Annexin 5 and 7 aad, by BioLegend (1 mentions) Mab1427, by R&D Systems (1 mentions)

15 protocols using adam10

Spike Protein Cleavage by Proteases

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Recombinant spike protein (SIN‐40589‐V08B1‐100, SinoBiologicals) was incubated with 1 µg of recombinant ADAM17 (Enzo Life Sciences) or ADAM10 (R&D) or TMPRSS2 (LSBio) for 24 h at 37°C at a 1:1 ratio. ADAM proteases were active in a 25 mM Tris buffer (2.5 µM ZnCl₂, 0.005% Brij‐35 (w/v), pH 9.0). For TMPRSS2, the reactions were performed in a 50 mM Tris buffer (0.2% Triton X‐100, 50 mM NaCl, pH 7.5). For inhibition of ADAMs and TMPRSS2, proteases were incubated with BB94 (10 µM) or camostat mesylate (100 µM), respectively, for 5 min before the addition of spike protein. Samples were boiled for 5 min at 95°C in a 2× Laemmli buffer and subjected to an 8% SDS‐PAGE. The cleavage fragments were detected using anti‐HIS (Clone 3D5) antibody.

Jocher G., Grass V., Tschirner S.K., Riepler L., Breimann S., Kaya T., Oelsner M., Hamad M.S., Hofmann L.I., Blobel C.P., Schmidt‐Weber C.B., Gokce O., Jakwerth C.A., Trimpert J., Kimpel J., Pichlmair A, & Lichtenthaler S.F. (2022). ADAM10 and ADAM17 promote SARS‐CoV‐2 cell entry and spike protein‐mediated lung cell fusion. EMBO Reports, 23(6), e54305.

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Enzymatic Characterization of Metalloprotease Inhibition

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The catalytic domain of TACE was produced in insect cells system as previously described28 (link); the catalytic domains of MMP7, MMP9 and MT1-MMP are all expressed and purified in our laboratory and human ADAM10 was purchased from R&D. The enzymatic activities were tested by hydrolysis processing of fluoregenic peptide by purified TACE (10 nM) (Mca-PLAQAV-Dpa-RSSSR, 10 μM) (R&D system), MMP9 (10 nM), MMP7 (10 nM) and MT1-MMP (10 nM) (Mca-PLGL-Dpa-AR, 10 μM) (R&D system) and ADAM10 (Mca-KPLGL-Dpa-AR, 10 μM) (R&D system) at 37 °C monitoring the increasing fluorescence intensity at λ_ex = 340 nm and λ_em = 340 nm. The inhibition assay was carried out in a range of concentration of TPD (3 nM–2500 nM) after pre-incubation in 37 °C for 30 min. Initial reaction rates were measured and inhibition constant were evaluated by fitting to equation of IC₅₀.

Wong E., Cohen T., Romi E., Levin M., Peleg Y., Arad U., Yaron A., Milla M.E, & Sagi I. (2016). Harnessing the natural inhibitory domain to control TNFα Converting Enzyme (TACE) activity in vivo. Scientific Reports, 6, 35598.

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Characterization of Matrix Metalloproteinases

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MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, ADAM10, ADAM17 and Mca-KPLGL-Dpa-AR-NH₂ fluorogenic peptide substrate were purchased from R&D Systems (cat # 901-MP, 902-MP, 908-MP, 911-MP, 910-MP, 511-MM, 918-MP, 936-AD, 930-ADB, and ES010, respectively). All common chemicals were purchased from Sigma. NFF449 was purchased from Tocris (cat# 1391) and actinonin was from Sigma-Aldrich, St. Louis, MO, USA (cat# 01809).

Wang C., Diez J., Park H., Becker-Pauly C., Fields G.B., Spicer T.P., Scampavia L.D., Minond D, & Bannister T.D. (2021). Discovery and Optimization of Selective Inhibitors of Meprin α (Part II). Pharmaceuticals, 14(3), 197.

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Multiparametric Immune Cell Profiling

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Anti-mouse CD16/CD32 (Clone 2.4G2), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), MHC II (M5/114.15.2), F4/80 (BM8), Clec4F (3E3F9), Tim4 (RMT4-54), CD11b (M1/70), Ly6C (HK1.4), TCR-β (H57-597), NK1.1 (PK136), CD4 (GK1.5), CD8 (53–6.7), CXCR6 (SA051D1), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD103 (2E7), IFN-γ (XMG1.2), as well as Annexin V and 7-AAD were purchased from Biolegend. Anti-mouse CRIg (NLA14), Nr4a1 (12.14) were purchased from ThermoFisher Scientific. CXCL16 (12–81) was purchased from BD Biosciences. ADAM10 (139712) was purchased from R&D Systems.

Liu G., Abas O., Strickland A.B., Chen Y, & Shi M. (2021). CXCR6+CD4+ T cells promote mortality during Trypanosoma brucei infection. PLoS Pathogens, 17(10), e1009968.

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Flow Cytometric Analysis of Cell Surface Receptors

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Cells were harvested with trypsin-EDTA (Lonza) and washed in FACS buffer (PBS containing 1% FBS). 5×10⁵ cells were stained in a volume of 25μl for 30 minutes at 4°C. Antibodies were diluted in FACS buffer using the following concentrations: FITC conjugated a-HER2 affibody (1:1500, Bromma, Sweden); a-EGFR (1:2000, clone H11, DAKO, Carpinteria, CA); a-HER3 (1:1500, clone SGP1, Abcam); a-HER4 (1:200, clone H4.77.16, Abcam); a-IGFR (1:50, clone 33255, R&D, Minneapolis, MN); ADAM10 (1:500, MAB1427, R&D); ADAM17 (1:100, MAB9301, R&D). Secondary APC labeled a-mouse (550826, BD) was diluted 1:800. After washing, cells were resuspended in FACS buffer containing 50ng/ml propidium Iodide (PI) (Sigma) and acquired on a FACSCanto II (BD, Franklin Lakes, NJ). Data were analyzed with FlowJo 10 (Tree Star, Ashland, OR). The geometric mean fluorescence (gMFI) intensity in the relevant channel was calculated from the PI negative gate; gMFI from the isotype control was subtracted from the sample yielding the delta gMFI.

Ebbing E.A., Medema J.P., Damhofer H., Meijer S.L., Krishnadath K.K., van Berge Henegouwen M.I., Bijlsma M.F, & van Laarhoven H.W. (2016). ADAM10-mediated release of heregulin confers resistance to trastuzumab by activating HER3. Oncotarget, 7(9), 10243-10254.

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Aged Dendritic Cell Cytokine Response

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PBECs were exposed to supernatants from aged and young DCs for 6 h. For
experiments in Figure 5,
recombinant TNF-α (100 pg ml ⁻¹) or a combination of
TNF-α, IL-6 (5 ng ml ⁻¹), CXCL-10 (5 ng ml
⁻¹; PeproTech), ADAM10 (1 μg ml
⁻¹; R&D Systems) was added to supernatants from young
DCs for 6 h. RNA was extracted using TRIZOL. Real-time PCR was performed using
validated specific primers for CD54, MUC-1, TLR2, chemokines (Real-time primers;
LLC, Elkins Park, PA). The data were normalized to β-actin. Relative
quantification method was used for calculations and data are expressed as fold
increase over controls.

Prakash S., Agrawal S., Vahed H., Ngyuen M., BenMohamed L., Gupta S, & Agrawal A. (2014). Dendritic cells from aged subjects contribute to chronic airway inflammation by activating bronchial epithelial cells under steady state. Mucosal immunology, 7(6), 1386-1394.

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ADAM10-mediated Fc-BCAM cleavage analysis

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One microgram of Fc‐BCAM was incubated with 500 ng of recombinant human ADAM10 (#936‐AD; R&D Systems) in activity buffer (1 mM ZnCl₂; 20 mM Tris–HCl pH 8,0; 10 mM CaCl₂; 150 mM NaCl; 0.0006% Brij‐35) for 5 h at 37°C in the presence or absence of the zinc chelator TPEN (50 μM). Samples were prepared for proteomic analysis by acetone precipitation, resolubilisation in 8 M Urea, reduction (10 mM DTT) and alkylation (55 mM iodoacetamide), followed by 2 h incubation with LysC (1:100; Wako Chemicals, Neuss, Germany), dilution to 2 M urea using 50 mM TEAB and overnight digestion using trypsin (1:50; Serva). After solid phase extraction on STAGE tips,³² LC/MS²‐analysis was performed as described²⁹ and data analysed with MaxQuant using the human Uniprot database (canonical and isoforms;194237 entries; downloaded 2021/02/08). The relevant instrument as well as MaxQuant parameters are extracted using MARMoSET and included in the supplementary material.

Sivakumar S., Lieber S., Librizzi D., Keber C., Sommerfeld L., Finkernagel F., Roth K., Reinartz S., Bartsch J.W., Graumann J., Müller‐Brüsselbach S, & Müller R. (2023). Basal cell adhesion molecule promotes metastasis‐associated processes in ovarian cancer. Clinical and Translational Medicine, 13(1), e1176.

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Optimized Enzymatic Assays for ADAM and MMP Proteins

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ADAM10, MMP13 and the fluorogenic peptide substrate IX ((7-Methoxycoumarin-4-yl)acetyl-Lys-Pro-Leu-Gly-Leu-N-3-(2,4-Dinitrophenyl)-L-2,3-diaminopropionyl-Ala-Arg-NH₂) were products of R&D Systems, MN. ADAM17 (residue 1–473), MMP14 and MMP19 were produced in house using Sf-9 insect cell or refolded from Escherichia coli inclusion bodies following published protocols [15 , 22 , 23 ]. Nickel sepharose excel resin was obtained from GE Healthcare Life Sciences, USA. Human TNF-α ELISA kit was a product of Sino-Biologic Inc., China. Mammalian cell lines were purchased from the Shanghai Cell Bank, Chinese Academy of Science.

Duan J.X., Rapti M., Tsigkou A, & Lee M.H. (2015). Expanding the Activity of Tissue Inhibitors of Metalloproteinase (TIMP)-1 against Surface-Anchored Metalloproteinases by the Replacement of Its C-Terminal Domain: Implications for Anti-Cancer Effects. PLoS ONE, 10(8), e0136384.

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Characterization of MMP and ADAM Enzymes

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MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, ADAM10, and ADAM17 were purchased from R&D Systems (cat # 901-MP, 902-MP, 908-MP, 911-MP, 910-MP, 511-MM, 918-MP, 936-AD, and 930-ADB, respectively). All common chemicals were purchased from Sigma. Marimastat was purchased from Tocris (cat# 2631), actinonin was from Sigma-Aldrich (cat# 01809). Hybricare medium was from ATCC (ATCC® 46-X™).

Madoux F., Dreymuller D., Pettiloud J.P., Santos R., Becker-Pauly C., Ludwig A., Fields G.B., Bannister T., Spicer T.P., Cudic M., Scampavia L.D, & Minond D. (2016). Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate. Scientific Reports, 6, 11.

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Proteolytic Activity of ADAM17 on IFN-γ

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Recombinant IFN-γ (1 μg; Wako) was incubated with intact or heat-inactivated recombinant ADAM17 (rADAM17) or ADAM10 (100 ng each; R&D Systems, Minneapolis, MN) in assay buffer (25 mM Tris, 2.5 μM ZnCl₂, 0.005% Brij-35, pH 9.0) for 1.5 h. Then NuPAGE LDS Sample Buffer (Life Technologies, Tokyo, Japan) containing reducing agent was added to each sample. After boiling, the protein samples were separated by Mini-PROTEAN Tris-Tricine gel (Bio-Rad Laboratories, Hercules, CA), electrophoresis, and stained with Bio-Safe Coomassie Brilliant Blue (Bio-Rad) or Silver Stain MS Kit (Wako). In some experiments, a protein band was excised and subjected to nano LC-MS/MS analysis for peptide sequence identification (Japan Proteomics, Sendai, Japan).
To observe the degradation activity of ADAM17 against other IFNs, recombinant human interferon β1a (ProSpec-Tany TechnoGene, Rehovot, Israel) was tested as a substrate.

Kanzaki H., Shinohara F., Suzuki M., Wada S., Miyamoto Y., Yamaguchi Y., Katsumata Y., Makihira S., Kawai T., Taubman M.A, & Nakamura Y. (2016). A-Disintegrin and Metalloproteinase (ADAM) 17 Enzymatically Degrades Interferon-gamma. Scientific Reports, 6, 32259.

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