Recombinant Human PKLR 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 PKLR (rhPKLR) (Catalog # 8569-PK)
• Activator: D-Fructose 1,6-bisphosphate trisodium salt hyrate (Sigma, Catalog # F6803), 50 mM stock in deionized water
• Adenosine 5’-diphosphate sodium salt (ADP) (Sigma, Catalog # A2754), 200 mM stock in diH2O
• Phospho(enol)pyruvic acid monosodium salt hydrate (PEP) (Sigma, Catalog # P0564), 50 mM stock in deionized water
• Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
• Kinase Glo® Plus Luminescent Kinase Assay (Promega, Catalog # V3771)
• 96-well Solid White Plate (Corning Life Sciences, Catalog # 3912)
• Plate Reader (Model: 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 rhPKLR to 0.25 µg/mL in Assay Buffer.
3. Dilute Activator to 0.133 mM in Assay Buffer.
4. Stimulate enzyme by combining 40 µL of 0.25 µg/mL rhPKLR and 60 µL of 0.133 mM Activator. Replace enzyme with Assay Buffer for a Control.
5. Incubate for 5 minutes at room temperature.
6. Prepare a substrate mixture of 5 mM ADP and 4 mM PEP in Assay Buffer.
7. Add 100 µL of the substrate mixture to incubated rhPKLR and Control. Mix briefly.
8. Incubate all at room temperature for 15 minutes.
9. Dilute ATP to 100 µM in Assay Buffer.  This is the first point of the standard curve. Complete the curve by performing six two-fold dilutions of 100 µM ATP in Assay Buffer. The standard curve has a range of 0.078 to 5.0 nmol per well.
10. Load 50 µL of each dilution of the standard curve in triplicate into a solid white plate. Include a curve blank containing 50 μL of Assay Buffer.
11. After incubation (step 8), heat reactions and control at 90-100 °C for 1 minute to stop the reaction. Cool on ice and centrifuge briefly to collect any condensation.
12. Load 50 µL of each reaction and control in triplicate into the same plate containing the standard curve.
13. Add 50 µL of Kinase Glo® Plus Reagent to all wells used, including standard curve.
14. Incubate at room temperature for 10 minutes in the dark.
15. Read plate in Luminescence endpoint mode.
16. Calculate specific activity:

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

• rhPKLR: 0.0025 µg
• ADP: 1.25 mM
• PEP: 1.0 mM
• Activator: 20 µM