Recombinant Human PKM2 Protein, CF

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

• Assay Buffer: 0.1 M MES, 5 mM MgCl₂, 20 mM KCl, pH 6.5
• Recombinant Human PKM2 (rhPKM2) (Catalog # 7244-PK)
• D-Fructose 1,6-bisphosphate trisodium salt hydrate (Sigma, Catalog # F6803), 50 mM stock in 50 mM Tris, pH 7.5
• Adenosine 5’-diphosphate sodium salt (ADP) (Sigma, Catalog # A2754), 200 mM stock in deionized water
• Phospho(enol)pyruvic acid monosodium salt hydrate (PEP) (Sigma, Catalog # P0564), 100 mM stock in deionized water
• Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
• Kinase-Glo® Plus Luminiscent Kinase Assay (Promega, Catalog # V3771)
• 96-well Solid White Plate (Corning Life Sciences, Catalog # 3912)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Follow the Kinase-Glo® Plus Luminescent Kinase Assay kit directions for preparing Kinase-Glo® Plus Reagent. Use the reagent immediately or freeze aliquots at -20 °C for future use. (Note: This assay measures an increasing luminescent signal, which is opposite of the traditional use of the kit).
2. Dilute rhPKM2 to 4 μg/mL in Assay Buffer.
3. Dilute D-Fructose 1,6-bisphosphate to 40 μM in Assay Buffer.
4. Stimulate enzyme by combining equal volumes of diluted rhPKM2 and D-Fructose 1,6-bisphosphate. As a control combine equal volumes of Assay Buffer and D-Fructose 1,6-bisphosphate.
5. Incubate for 5 minutes at room temperature.
6. Dilute incubated rhPKM2 to 0.2 µg/mL in Assay Buffer. Perform an identical dilution to the control.
7. Prepare a substrate mixture of 5 mM ADP and 5 mM PEP in Assay Buffer.
8. Combine 80 µL of diluted rhPKM2 with 80 µL of the substrate mixture in microcentrifuge tubes. Also prepare a Blank by combining 80 µL of diluted control with 80 µL of substrate mixture. Mix briefly.
9. Incubate at room temperature for 15 minutes.
10. Dilute ATP to 100 µM in Assay Buffer. Perform six two-fold dilutions in Assay Buffer. The standard curve has a range of 0.078‑5.0 nmol per well.
11. Load 50 µL of each dilution of the standard curve into a plate. Include a Curve Blank containing 50 μL of Assay Buffer.
12. Heat tubes to 95-100 °C for 3 minutes to stop the reaction. Cool on ice for 3 minutes and centrifuge tubes briefly to collect any condensation. Caution: overheating may lead to degradation of ATP produced.
13. Load plate with 50 µL of each reaction, in triplicate.
14. Add 50 uL of Kinase-Glo® Plus Reagent to all wells used. Incubate at room temperature for 10 minutes.
15. Read plate in Luminescence endpoint mode.
16. Calculate specific activity:

     *Adjusted for Blank
     **Derived from the ATP standard curve using linear fitting and adjusted for Curve Blank.

• rhPKM2: 0.005 µg
• ADP: 1.25 mM
• PEP: 1.25 mM
• ATP standard curve: 0.078-5.0 nmol