Thrombin activity was measured as previously described [29 (link)]. In brief, after perfusion with 4 ℃ PBS, the injured spinal cord epicenter was isolated and cut into 0.5 cm (20–30 mg). Epicenters were homogenized by assay buffer without the substrate (50 mM Tris·HCl, pH 5.8, 150 mM NaCl, 1 mM CaCl2, 0.1% BSA). The homogenate was centrifuged at 12000g for 13 min at room temperature. Each 50 μL sample was mixed with 150 μL assay buffer mixed with the substrate (protease inhibitor Prolyl Endopeptidase Inhibitor II (20 mM), Benzoyl-Phe-Val-Arg-AMC·HCl (13 mM)). The mixture was added to a 96-well black microplate. The kinetic fluorescence was determined for 45 min at 27 ℃ by a fluorescence detection system (SYNERGY, BioTek; excitation 360/40 nm, emission 450/40 nm). Thrombin substrate Benzoyl-Phe-Val-Arg-AMC·HCl (B7632) and Prolyl Endopeptidase Inhibitor II (537011) were purchased from Merck (Germany).
Prolyl endopeptidase inhibitor 2
Manufactured by Merck Group
Sourced in Germany
Prolyl Endopeptidase Inhibitor II is a laboratory reagent used to inhibit the activity of prolyl endopeptidase, an enzyme involved in the hydrolysis of peptide bonds. This product provides a tool for researchers to study the role of prolyl endopeptidase in various biological processes.
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Lab products found in correlation
Black 96 well microplate, by Thermo Fisher Scientific (1 mentions)
Infinite 200, by Tecan (1 mentions)
T4648, by Merck Group (1 mentions)
Rabbit anti map2 antibody, by Merck Group (1 mentions)
β actin monoclonal antibody, by Merck Group (1 mentions)
Fluorescein isothiocyanate fitc dextran, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Argatroban, by Enzo Life Sciences (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Micro bca kit, by Beyotime (1 mentions)
Bestatin hydrochloride, by Merck Group (1 mentions)
4 protocols using prolyl endopeptidase inhibitor 2
1
Quantifying Spinal Cord Thrombin Activity
2
Quantifying Thrombin Activity in Tissue
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Thrombin activity was assessed using a fluorogenic thrombin substrate (Bachem I-1560, excitation 360 nm; emission 465 nm), as previously described [18 (link)]. Human biopsies were thawed and washed 3 times for 5 min in Tris-buffer (contains in mM: 150 NaCl, 1 CaCl2, 50 Tris-HCl: pH 8.0) on ice. Human and rodent biopsies were placed in a black 96-well microplate (Nunc, Roskilde, Denmark) containing Tris-buffer. The plate was incubated at 37 °C for 30 min before the addition of the substrate. To eliminate the effect of abundant endopeptidases in the assay, endopeptidase inhibitors (0.1 mg/mL, bestatin hydrochloride—B8385, Sigma; 200 μM prolyl endopeptidase inhibitor II, 537011, Merck Millipore) were added to the substrate. Known bovine thrombin concentrations (T-4648, Sigma) were used to create a calibration curve for each experiment. The specific thrombin inhibitor SIXAC (100 nM, American Peptide Company, Sunnyvale, CA, USA) [19 (link)] was added to chosen wells to assess the specificity of the assay. The cleavage of the substrate was measured using a microplate reader (Tecan; infinite 200; Männedorf, Switzerland). Each biopsy was weighed, and the measured thrombin activity was normalized to tissue weight. The results are presented as mU thrombin activity/mg of tissue or relative to the control.
3
Inhibitors and Antibodies for Protease Research
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Nafamostat mesilate (C19H17N5O2.2CH403S, 6-amino-2-naphthyl p-guanidinobenzoate dimethanesulfonate), a serine protease inhibitor, was obtained from NANJING D&R PHARMACEUTICAL COMPANY (China). Argatroban (C23H36N6O5S•H2O, 1-[5-[(aminoiminomethyl)amino]-1-oxo-2 -[[(1,2,3,4-tetrahydro-3-methyl-8-quinolinyl)sulfonyl]amino]pentyl]-4-methyl-2-piperidinecarboxylic acid) was purchased from Enzo Life Sciences (BML-PI146, USA). Fluorescein isothiocyanate (FITC) dextran was purchased from Sigma-Aldrich (USA), goat anti-thrombin antibody was purchased from Santa Cruz Biotechnology (USA), rabbit anti-MAP-2 antibody was purchased from Chemicon (USA), β-actin monoclonal antibody was purchased from Sigma-Aldrich (USA), Alexa Fluo-488 conjugated goat anti-rabbit IgG antibody and Alexa Fluo-594 conjugated donkey anti-goat IgG antibody were purchased from Invitrogen (CA). RIPA buffer and MicroBCA kit were purchased from Beyotime (China). Protease inhibitor cocktail was purchased from Roche (Indianapolis, IN, USA). Thrombin substrate Benzoyl-Phe-Val-Arg-AMC·HCl and Prolyl Endopeptidase Inhibitor II were purchased from Merck (Germany).
4
Quantifying Thrombin Activity in CSF
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Thrombin activity was assessed using a fluorogenic substrate cleaved by thrombin (Cat# I-1560; Bachem, Bubendorf, Switzerland; excitation 360 nm; emission 465 nm). The reactions were carried out in a black 96-well microplate using 60 μL of CSF. All reactions were performed in Tris buffer (in mM: 150 NaCl, 1 CaCl2, 50 Tris-HCl; pH 8.0). In order to exclude the effect of widely abundant CNS endopeptidases, all reactions were performed in the presence of endopeptidase inhibitors [bestatin hydrochloride (0.1 mg/mL), Cat# B8385, Sigma, St. Louis, MO, USA; 200 μM prolylendopeptidase inhibitor II, Cat# 537011, Merck Millipore, Burlington, MA, USA]. Controlled reactions were performed in each experiment in the presence of N-alpha-((2-naphthylsulfinyl) glycyl)-DL-p-amidinophenylalanylpiperidine (NAPAP), a specific thrombin inhibitor (1 μM; Cat# SC-208083; Santa Cruz Biotechnology, Dallas, TX, USA) to evaluate the specific contribution of thrombin to substrate cleavage. Calibration curve was created for each experiment using known bovine thrombin concentrations (Cat# T-4648, Sigma). Thrombin activity was normalized to total CSF protein concentration, which was measured by the Pyrogallol Red Molybdate protein dye-binding assay using Beckman Coulter au5822 (Beckman Instruments Inc., Fullerton, CA, USA).
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