Recombinant Human PSAT1 His-tag Protein, CF

• Assay Buffer: 50 mM Tris, 16 mM Ammonium Acetate, pH 8.5
• Recombinant Human PSAT1 (rhPSAT1) (Catalog # 10342-PS)
• Recombinant Human PHGDH (rhPHGDH) (Catalog # 10131-DH)
• O-Phospho-L-serine (Tocris, (Catalog # 0238) 100 mM stock in 20 mM Tris, pH 8.5
  
• beta -Nicotinamide Adenine Dinucleotide, reduced (NADH) (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
• alpha -Ketoglutaric acid (Sigma, Catalog # K2010), 1 M stock in deionized water
• Pyridoxal 5'-phosphate (Sigma, Catalog # P9255), 100 mM stock in 1 M HEPES, pH 8.0
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare a substrate mixture containing 0.4 mM NADH, 2 mM alpha -Ketoglutaric acid, 40 µM Pyridoxal 5'-phosphate, 2 mM O-Phospho-L-serine and 14 µg/mL rhPHGDH in Assay Buffer. Mix well and incubate at room temperature for 5 minutes.
2. Dilute rhPSAT-1 to 20 µg/mL in Assay Buffer.
3. Load 50 µL of 20 µg/mL rhPSAT-1 into wells of a plate, and start the reaction by adding 50 µL of substrate mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of substrate mixture.
4. Read plate at an absorbance of 340 nm in kinetic mode for 10 minutes with a lag time of 3 minutes.
5. Calculate specific activity:
    
   

Per Well:

• rhPSAT-1: 1.0 µg
• O-Phospho-L-serine: 1 mM
• NADH: 0.2 mM
• alpha -Ketoglutaric acid: 1 mM
• Pyridoxal 5'-phosphate: 20 µM
• rhPHGDH: 0.7 µg