Recombinant S. marcescens NucA nuclease Protein, CF

Recombinant S. marcescens NucA nuclease Protein, CF (Catalog # 10038-NAB) from R&D Systems and a competitor have similar Specific Activity (pmol/min/µg) measured by its ability to hydrolyze DNA from salmon testes in direct side-by-side comparison using the insert assay protocol described.

2 μg/lane of Recombinant S. marcescens NucA nuclease Protein (Catalog # 10038-NAB) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 27-30 kDa, under reducing conditions.

Recombinant S. marcescens NucA nuclease Protein, CF (Catalog # 10038-NAB) purity was determined by HPLC.

• Reactivity: Ba
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant S. marcescens NucA nuclease Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze DNA from salmon testes. The specific activity is >150,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived s. marcescens NucA nuclease protein
  Asp22-Asn266
• Accession #: P13717.2
• N-terminal Sequence: Asp22
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >97%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 27 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 27-30 kDa, under reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >97%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 100 mM Tris, 1 mM MgCl₂, pH 8.5
  • Recombinant S. marcescens NucA (rS.m.NucA) (Catalog # 10038-NAB)
  • Deoxyribonucleic acid (DNA) sodium salt from salmon testes, 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 with Absorbance Read Capability
  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.m.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:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

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

  Per Reaction:
  • rS.m.NucA: 0.1 ng (0.0001 µg)
  • rmALPL: 0.1 µg
  • DNA: 4 µg

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant S. marcescens NucA nuclease Protein, CF

• DcD
• NucA nuclease

Background

Serratia marcescens endonuclease NucA is a non-specific nuclease that degrades both single- and double-stranded nucleic acids, including DNA and RNA, but exhibits no proteolytic activity (1) making it ideal for the removal of nucleic acid contaminants from protein samples and in applications where complete digestion of nucleic acids is desirable. NucA is commonly used in bioprocessing applications to reduce viscosity of samples caused by genomic DNA. NucA hydrolyzes internal phosphodiester bonds between nucleotides in nucleic acids to produce 5'-monophosphate oligonucleotides of 3-8 bases in length (2). The active enzyme is a homodimer with two disulfide bonds in each monomer that are crucial to the enzyme activity and stability (3). The optimum pH for enzyme activity is found to be 8.0-9.2. The activity of the recombinant enzyme is measured by a phosphatase coupled assay (4), where the 5'-phosphate of oligosaccharides generated by the enzyme is further released by non-specific alkaline phosphatase and quantitated by Malachite reagents (5).

1. Benedik, MJ and Strych, U. (1998) FEMS Microbiol. Lett. 165:1.
2. Nestle, M, et al. (1999) J. Biol. Chem. 274:825.
3. Ball, T.K. et al. (1992) Nucleic Acids Res. 20:4971.
4. Wu, Z.L. et al. (2011) Glycobiology 21:727.
5. Van Veldhoven, P.P. and G.P. Mannaerts (1987) Anal. Biochem. 161:45.

Bioinformatics

• Uniprot:
  • P13717.2()