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Protocol

COX Assay
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Enzymes; Cox I and Cox II Cox I; from Sigma; Total 5,000 units/250 microl (0.3 mg protein; 58116 u/mg) Prep; 5,000 units/ml (Phosphate buffer 100mM, pH = 7.5); 500 units/ml used as enzyme Stored at -70 C Stable for 2-8 hours in ice Cox II; from Sigma; Total 1,000 units/160 microl (0.57 mg protein; 11034 u/mg) Prep; 1,000 units/ml (Phosphate buffer 100 mM, pH = 7.5); 330 units/ml used as enzyme Stored at -70 C Stable 2-8 hours in ice Phosphate buffer; 100 mM Na2HPO4, adjust pH = 7.5. Hematin; Prepare stock hematin by dissolving in DMSO at 0.380 mg/ml, and dilute for working hematin in 100mM Phosphate buffer, pH = 7.5 to a final concentration of 0.12 microM/L (stable for up to 8 hours at room temperature). Arachidonic acid; Under argon or nitrogen, add ethanol to arachidonic acid (50 Mg/ml); store at -70 C. To activate add 94 microl of 0.1 N sodium hydroxide and 6 microl of arachidonic acid prepared above (50 mg/nl). Vortex and dilute with 9.9 ml of deionized water. Stable for up to 3 hours in ice. Tris-Phenol Buffer; 100 mM Tris, 0.5 mM phenol buffer, pH = 7.3.
Assay 1. Add 50 l of the tris-phenol buffer into positions of the 96-well plate. 2. Add 50 l of the hematin solution. 3. Add 50 microl of Cox I or Cox II (total = 20 units/275 microl). 4. Incubate for 5 minutes at room temperature. 5. Add 25 microl of inhibitor solution (use 300 mM Ibuprofen for Cox I, And 1 mM of NS-398 for Cox II). 6. Incubate for from 5-120 minutes (dependent on inhibition) at room temperature (did 10 min). 7. Place the plate into the luminometer. 8. Inject 50 microl of COX substrate using injector 1 (P). 9. Immediately inject 50 microl of the activated arachidonic acid using injector 2 (M). 10. Read for % sec for relative light units (RLU) output.
Instrument Settings: Delay inject P/Measurement Interval 1: 1.6 seconds Measurement Time Interval 1: 0 seconds Delay inject M/Measurement Interval 2: 1.6 seconds Measurement Time Interval 2: 5.0 seconds Injector P (1): 50 microL of Cox substrate Injector M (2): 50 microL of Activated Arachidonic acid Sample (or inhibitor) volumes: 25 microL

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