Recombinant S. marcescens NucA nuclease Protein, CF

• Assay Buffer: 100 mM Tris, 1 mM MgCl₂, pH 8.5
• rS.marcescens NucA (Catalog # 10038-NA)
• Deoxyribonucleic acid (DNA) sodium salt from salmon testes (Millipore Sigma, Catalog # D1626), 5 mg/mL stock in deionized water
• Coupling Enzyme: Recombinant Mouse Alkaline Phosphatase/ALPL (rmALPL) (Catalog # 2910-AP)
• 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 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. Complete the 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 nmol per well.
3. Prepare a reaction mixture containing 0.16 mg/mL DNA and 4 µg/mL rmALPL in Assay Buffer.
4. Dilute rS. marcescens NucA to 4 ng/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 4 ng/mL rS. marcescens NucA into empty wells of the same plate as the curve. Include a Control containing 25 µL of Assay Buffer.
7. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
8. Seal 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.

• rS. marcescens NucA: 0.1 ng (0.0001 µg)
• rmALPL: 0.1 µg
• DNA: 4 µg

Scientific Data

Enzyme Activity View Larger

2 μg plasmid DNA was incubated with increasing amounts (ng/mL) of RecombinantS. marcescensNucA nuclease (Catalog # 10038-NA) in 50 mM Tris (pH 8.0) and 1 mM MgCl2 for 30 minutes at room temperature. Samples were analyzed by agarose gel electrophoresis and DNA was visualized by ethidium bromide staining.