Recombinant Human ODC1 His-tag Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM Tris, pH 7.5
• Enzyme Buffer: 50 mM Tris, 0.1 mM EDTA, 0.1 mM Pyridoxal 5'-phosphate, 2.5 mM DTT, 0.1% Tween 80, pH 7.5
• Recombinant Human Ornithine Decarboxylase 1 (rhODC1) (Catalog # 10316-OD)
• Substrate: L-Ornithine monohydrochloride (Sigma, Catalog # O2375), 1 M stock in deionized water
• Cucurbit[6]uril Hydrate (CB6) (Sigma, Catalog # 94544), 150 µM stock in 50 mM Tris and 1 M HCl
• Trans-4-[4-(Dimethylamino)styryl]-1-methylpyridinium iodide (DSMI) (Sigma, Catalog # 336408), 40 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax M5 by Molecular Devices) or equivalent

1. Dilute CB6 and DSMI to 0.667 µM and 4 µM in Assay Buffer, respectively.
2. In a plate load 150 µL of diluted CB6/DSMI mixture and incubate for 10 minutes at room temperature in the dark.
3. Dilute rhODC1 to 0.1 µg/mL in Enzyme Buffer.
4. Dilute Substrate to 200 µM in Assay Buffer.
5. Add 25 µL of 0.1 µg/mL rhODC1 to wells containing the CB6/DSMI mixture, and start the reaction by adding 25 µL of 200 µM Substrate. Include a Substrate Blank containing 25 µL of Enzyme Buffer and 25 µL of 200 µM Substrate (along with 150 µL of CB6/DSMI mixture).
6. Read at excitation and emission wavelengths of 450 nm and 582 nm (top read), respectively, in kinetic mode for 5 minutes.
7. Calculate specific activity:

*Adjusted for Substrate Blank
**Derived using calibration standard putrescine (Sigma, Catalog # 51799) in the presence of the CB6/DSMI mixture.

• rhODC1: 0.0025 µg
• CB6: 0.5 µM
• DSMI: 3 µM
• Substrate: 25 µM