Recombinant Human MAT2A 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, 50 mM KCl, 10 mM MgCl₂, 0.05% Brij-35, pH 8.0
• Recombinant Human Methionine Adenosyltransferase 2A (rhMAT2A) (Catalog # 10315-MA)
• L-Methionine (Sigma, Catalog M5308), 300 mM stock in deionized water
• ATP (Sigma, Catalog # A7699), 400 mM stock in deionized water
• Malachite Green Phosphate Detection Kit (Catalog # DY996)
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 M Phosphate Standard provided by the Malachite Green Phosphate Detection Kit by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for a 10 mM stock. Continue by adding 10 µL of the 10 mM phosphate stock to 990 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
   
2. Continue standard curve by performing six one-half serial dilutions of the 100 µM phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmoles per well.
3. Prepare Reaction Mixture containing 400 µM ATP and 600 µM L-Methionine in Assay Buffer.
4. Dilute rhMAT2A to 20 µg/mL in Assay Buffer.
5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
6. Load 25 µL of the 20 µg/mL rhMAT2A into empty wells of the plate. Include a Control containing 25 µL of Assay Buffer.
7. Start the reactions by adding 25 µL of Reaction Mixture to all the wells, excluding the standard curve and curve blank.
8. Seal the plate and incubate at room temperature for 20 minutes.
9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
10. Add 100 µL of deionized water to all wells. Mix briefly.
11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
12. Read plate at 620 nm (absorbance) in endpoint mode.
    
13. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

• rhMAT2A: 0.5 µg
• ATP: 200 µM
• L-Methionine: 300 µM