Recombinant E. coli BirA His-tag Protein, CF

• Assay Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5 
• Recombinant E. coli BirA His-tag (rEBirA) (Catalog # 10464-BA)
• Recombinant Yeast Inorganic Pyrophosphatase/PPA1 (ryPPA1) (Catalog # 8088-PP)
• Recombinant Human Tumor Necrosis Factor Receptor Superfamily Member 4 (rhOX40)
• Magnesium Chloride (Sigma, Catalog # M8266), 1 M stock in deionized water
• Biotin (Thermo, Catalog # 29129), 10 mM stock in 5% (v/v) DMSO
• Adenosine triphosphate (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 by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water 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 using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
3. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing the 50 µL of Assay Buffer.
4. Prepare reaction mixture containing 2 mM ATP, 20 mM MgCl2, 800 µM Biotin, 280 µg/mL rhOX40, and 8 µg/mL ryPPA1 in Assay Buffer.
5. Dilute rEBirA to 40 µg/mL in Assay Buffer.
6. Load 25 µL of the 40 µg/mL rEBirA into empty wells of the same plate as the curve.  Include a Control containing 25 µL of Assay Buffer.
7. Load 25 µL of the reaction mixture to wells, excluding the standard curve.
8. Seal plate and incubate at room temperature for 10 minutes.
9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
10. Add 100 µL of the deionized water to all wells. Mix briefly.
11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate at room temperature for 20 minutes.
12. Read plate at 620 nm (absorbance) in endpoint mode. 
13. Calculate specific activity:

*Derived from the phosphate standard curve using linear fitting and adjusted for Control.

• rEBirA: 1 µg
• ryPPA1: 0.2 µg
• ATP: 1 mM
• rhOX40: 7 µg
• MgCl2: 10 mM
• Biotin: 400 µM

Scientific Data

SDS-PAGE View Larger

2 μg/lane of RecombinantE. coliBirA His-tag (Catalog # 10464-BA) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 30-38 kDa.