For activity of MMP-9 (gelatinase B) analysis, microglia conditioned medium was mixed with sample buffer and electrophoresed in 10 % SDS gels containing as substrate procaine gelatine. The detailed procedure was described previously4 (link). Briefly, after electrophoresis, gels were washed in a solution of 2.5% Triton X-100, and incubated overnight at 37 °C in developing buffer. Gels were then stained with Coomassie Brilliant Blue R250 and destained in a solution of 10 % acetic acid and 40 % methanol. Recombinant human MMP-9 (R&D systems) was used as a positive control.
Recombinant human mmp 9
Manufactured by R&D Systems
Sourced in United Kingdom, United States
Recombinant human MMP-9 is a laboratory reagent produced by R&D Systems. MMP-9, also known as matrix metalloproteinase-9, is a zinc-dependent endopeptidase that belongs to the matrix metalloproteinase (MMP) family. This recombinant protein is expressed in insect cells.
Automatically generated - may contain errors
4 protocols using recombinant human mmp 9
1
Quantitative Gelatinase B Activity Assay
2
Quantification of MMP-2 and MMP-9 Release
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Solutions (6 ng/ml) were reconstituted from Recombinant Human MMP-2 (R&D Systems, Abingdon, UK) and Recombinant Human MMP-9 (R&D Systems) in the following buffers: MMP-2 (20 mM Tris–HCL, 150 mM NaCl, 5 mM CaCl2 and 0.05% (w/v) Brij35, pH 7.4) and MMP-9 (50 mM Tris–HCL, 150 mM NaCl, 10 mM CaCl2 and 0.05% (w/v) Brij-35, pH 7.5). A 5 mm piece of each dressing was soaked with 1 ml of each solution and incubated at 37 °C at 105 rpm for 4 days. After this incubation, supernatants were collected and ELISAs were performed according to the manufacturer’s instructions (Quantikine ELISA Total MMP-2 and Quantikine ELISA Human MMP-9—all R&D Systems) were imaged using a VersaMax microplate reader (Molecular Devices, San Jose, CA).
3
In Vitro VP Release Kinetics
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VP-TGMS samples (VP, 0.3 mg/mL, 200 μL) were suspended in PBS (10 mL, pH = 7.4) in a 15-mL centrifuge tube with or without recombinant human MMP-2 (100 ng/mL, R&D Systems, USA), recombinant human MMP-9 (15 ng/mL, R&D Systems, USA) or lipase (500 U/mL or 1000 U/mL, Aspergillus oryzae, Sigma–Aldrich, USA). Under certain conditions, the MMP-2/9 inhibitor ilomastat (10 nM, MedChemExpress, USA) was added together with MMP-2/9. The tubes were incubated at 37 °C in a humidified atmosphere. Fresh enzyme or inhibitor was added to the PBS solution at the determined time points. An aliquot (100 μL) was removed from the release solution, and the same volume of fresh PBS was replenished to ensure a constant volume. Collected aliquots were frozen, lyophilized, and resuspended in DMSO followed by fluorescence intensity detection. The mass of the VP released was back-calculated using standard curve.
4
Activation and Inhibition of Recombinant MMP9
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