Recombinant Human PGK-1 Protein, CF

• Assay Diluent: deionized water
• Recombinant Human PGK1 (rhPGK1) (Catalog # 5455-PK)
• 50 mM KH₂PO₄, pH 7.0
• 100 mM MgSO₄ in deionized water
• 1.0 M Glycine in deionized water
• 50 mM DL-Glyceraldehyde 3-Phosphate (GAP) (Sigma, Catalog # G5251) in deionized water
• 10 mM beta -Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522). Prepare 200 mM stock in deionized water
• 10 mM Adenosine 5’-Diphosphate (ADP) (Sigma, Catalog # A2754). Prepare 200 mM stock in deionized water. Note: ADP degrades to AMP which is an inhibitor of rhPGK1. Be sure to aliquot and store the stock at ≤-20 °C. Prepare fresh when necessary.
• 0.25 µg/µL Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) (Sigma, Catalog # G5537) in 50% Glycerol
• UV Plate, 96 well (Costar, Catalog # 3635)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare substrate buffer by mixing the following (prepare fresh):
1. 100 µL 50 mM KH₂PO₄, pH 7.0
2. 20 µL 50 mM GAP
3. 30 µL 10 mM beta -NAD
4. 20 µL 10 mM ADP
5. 50 µL 100 mM MgSO₄
6. 100 µL 1 M Glycine
7. 20 µL 0.25 µg/µL GAPDH
8. 160 µL deionized water

Note: This amount will assay nine wells. If more volume is needed, multiply each component’s volume by the same number to get the desired amount.
2. Dilute rhPGK1 to 0.02 ng/µL in deionized water.
3. Load in a 96 well UV plate 50 µL of the substrate buffer, and start the reaction by adding 50 µL of 0.02 ng/µL rhPGK1. Include a blank containing 50 µL of the substrate buffer and 50 µL deionized water.
4. Read at 339 nm in kinetic mode for 5 minutes.
5. 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

• rhPGK1: 0.001 μg
• Rxn mix: 5 mM KH₂PO₄, pH 7.0, 1 mM GAP, 0.3 mM beta -NAD, 0.2 mM ADP, 5 mM MgSO4, 100 mM Glycine, 5 ng/µL GAPDH