Recombinant Human Enolase 2 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, 30 mM MgCl₂, 200 mM KCl, pH 7.5
• Recombinant Human Enolase 2/Neuron‑specific Enolase His-tag (rhENO-2) (Catalog # 10412-EN).
• Substrate: L-2-Phosphoglyceric acid (2-PG) (Sigma, Catalog # 19710), 100 mM stock in deionized water
• Recombinant Human PKM-2 (PKM-2) (Catalog # 7244-PK)
• Recombinant Human LDH-A (LDH-A) (Catalog # 9158-HA)
• Adenosine diphosphate (ADP) (Sigma, Catalog # A2754), 200 mM stock in deionized water
• beta -Nicotinamide adenine dinucleotide, reduced disodium salt hydrate ( beta -NADH) (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
• 96-well clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare Reaction Mixture containing 20 µg/mL rhPKM-2, 5 µg/mL rhLDH-A, 14 mM ADP, 800 µM beta -NADH, and 8 mM 2-PG in Assay Buffer.
2. Incubate Reaction Mixture at room temperature for 5 minutes.
3. Dilute rhENO-2 to 1 µg/mL in Assay Buffer.
4. In a plate, load 50 µL of 1 µg/mL rhENO-2, and start the reaction by adding 50 µL of Reaction Mixture. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Reaction Mixture.
5. Read at an absorbance of 340 nm in kinetic mode for 10 minutes with a lag time of 3 minutes.
6. Calculate specific activity:

*Adjusted for Substrate Blank
**Using the extinction coefficient 6220 M^-1cm^-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD

• rhENO-2: 0.05 µg
• 2-PG: 4 mM
• ADP: 7 mM
• beta -NADH: 400 µM