To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fc-chimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl2, 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio). Controls included MMP-9 or target proteins alone in assay buffer. ADAM10 cleavage assays were performed in 25 mM tris-HCl (pH 7.4), 2.5 μM ZnCl2, and 0.05% Brij35. Samples were incubated for 16 hours at 37°C; reactions were stopped by the addition of Laemmli buffer (β-mercaptoethanol); samples were separated on 10% SDS-PAGE gels, and protein bands were detected by silver staining.
Recombinant mouse adam10
Manufactured by R&D Systems
Recombinant mouse ADAM10 is a laboratory product produced by R&D Systems. ADAM10 is a member of the ADAM (a disintegrin and metalloprotease) family of proteins. It functions as a metalloproteinase, capable of cleaving cell surface proteins.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant rat agrin, by R&D Systems (1 mentions)
Synergy h1 hybrid reader, by Agilent Technologies (1 mentions)
Gi 254023x, by R&D Systems (1 mentions)
Proteome profiler mouse cytokine array panel a, by R&D Systems (1 mentions)
Human timp 1 quantikine elisa kit, by R&D Systems (1 mentions)
Mca plaqav dpa rsssr nh2, by R&D Systems (1 mentions)
2 protocols using recombinant mouse adam10
1
In vitro Cleavage Assay of Cell Adhesion Proteins
2
Cytokine and TIMP1 Analysis in CL&P Mice
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For examination of cytokines in the serum of sham and CL&P mice, serum samples collected from five mice at 10 wk after sham or CL&P surgery were pooled, respectively. Cytokines were detected using Proteome Profiler Mouse Cytokine Array Panel A according to the manufacturer’s instructions (R&D).
For measurement of circulating TIMP1 levels, mouse serum was collected at indicated time points and human samples were obtained from septic patients and septic survivors. The amounts of TIMP1 were measured using a mouse or human TIMP1 Quantikine ELISA Kit according to the manufacturer’s instructions (R&D).
For evaluation of the inhibitory activity of TIMP1 on ADAM10, a colorimetric assay was performed to determine ADAM10-mediated cleavage of a fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH2 (R&D) in the presence or absence of TIMP1.
38 μM of recombinant mouse ADAM10 (R&D) was mixed with 20 μM of Mca-PLAQAV-Dpa-RSSSR-NH2 with or without 45 μM recombinant mouse TIMP1 (BioLegend) or 1 μM of the ADAM10 inhibitor GI 254023X (R&D). The reactions were incubated at 37°C for 30 min, and the amount of cleaved peptide substrate was measured by OD405 using a Synergy H1 Hybrid reader (BioTek) and was calculated as the relative unit of ADAM10 proteolytic activity.
For measurement of circulating TIMP1 levels, mouse serum was collected at indicated time points and human samples were obtained from septic patients and septic survivors. The amounts of TIMP1 were measured using a mouse or human TIMP1 Quantikine ELISA Kit according to the manufacturer’s instructions (R&D).
For evaluation of the inhibitory activity of TIMP1 on ADAM10, a colorimetric assay was performed to determine ADAM10-mediated cleavage of a fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH2 (R&D) in the presence or absence of TIMP1.
38 μM of recombinant mouse ADAM10 (R&D) was mixed with 20 μM of Mca-PLAQAV-Dpa-RSSSR-NH2 with or without 45 μM recombinant mouse TIMP1 (BioLegend) or 1 μM of the ADAM10 inhibitor GI 254023X (R&D). The reactions were incubated at 37°C for 30 min, and the amount of cleaved peptide substrate was measured by OD405 using a Synergy H1 Hybrid reader (BioTek) and was calculated as the relative unit of ADAM10 proteolytic activity.
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