Recombinant Human TIGAR/C12orf5 His-tag Protein, CF

• Assay Buffer: 50 mM Tris, pH 7.5
• Recombinant Human TIGAR/C12orf5 (rhTIGAR) (Catalog # 11008-TG)
• Substrate: 4-Nitrophenyl Phosphate (Sigma, Catalog # N2765), 10 mM stock in deionized water
• Sodium Hydroxide (NaOH) (Sigma, Catalog # 221465), 2 M stock in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhTIGAR to 40 µg/mL in Assay Buffer.
2. Dilute Substrate to 5 mM in Assay Buffer.
3. Dilute 2 M NaOH to 0.2 M NaOH in deionized water.
4. Prepare reaction mixtures by combining equivalent volumes of dilute rhTIGAR and dilute Substrate in microtubes. Include an Enzyme Control by combining dilute rhTIGAR with twice the volume of 0.2 M NaOH, mix briefly, then add a volume of dilute Substrate equivalent to the volume of rhTIGAR. The Enzyme Control will have 2x the volume of the reaction mixture.
5. Incubate Reactions and Enzyme Controls at 37 °C for 3 hours.
6. Load 100 µL of Reactions into a plate and stop the reactions by adding 100 µL of 0.2 M NaOH.
7. Load 200 µL of Enzyme Controls into plate.
8. Read plate at 410 (absorbance) in endpoint mode.
9. Calculate specific activity:
   

*Adjusted for Enzyme Controls
**Derived using calibration standard 4-Nitrophenol (Sigma, Catalog # 241326)

Per Well:

• rhTIGAR: 2 µg
• Substrate: 1.25 mM