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BenzNuclease DNA and RNA Nuclease(Ultra Pure,Protease Free)

  • BenzNuclease Enzyme Features
    1. Ideal for DNA and RNA clearance

    2. Free of detectable proteolytic activity

    3. Robust over a wide range of operating conditions

  • Description
    BenzNuclease is a recombinant form of Serratia macescens extracellular endonuclease produced in Escherichia coli cells using a proprietary process at ACRObiosystems. BenzNuclease is a homodimer with monomer molecular masses about 30 kDa. Two disulfide bonds found in the nuclease are crucial to its activity and stability. The enzyme is a non-specific nuclease with high specific activity, which degrades both single- and double-stranded nucleic acids in any form ( single stranded, double stranded, linear, circular and supercoiled ). It hydrolyzes internal phosphodiester bonds present between the nucleotides to 5'-phosphorylated oligonucleotides of 3-8 bases in length.
  • Application
    Its high intrinsic activity and broad substrate tolerance make the endonuclease an ideal tool in a variety of biotechnological and pharmaceutical applications: removal of nucleic acid from protein samples ( Elimination of nucleic acids from recombinant proteins; Purification of protein fragments from inclusion bodies; Sample preparation in western blotting or two-dimensional gel electrophoresis) ; Viscosity reduction in protein extracts.
  • Effect of Conditions on Activity
    BenzNuclease is functional between pH 6 and 10 (optimal at pH8 - 8.5) , and from 0℃ to 42 ℃ (optimal at 35 ℃ - 42 ℃). Mg2+ (1-2 mM) is required for enzyme activity.  1 mM EDTA reduced the activity by 30% in the presence of 1 mM MgCl2; 0.1 M EDTA eliminated all enzyme activity. In the presence of 1 mM MgCl2, enzyme levels were reduced 75% by 0.1 M CaCl2 or 1 M NaCl. Under standard assay conditions, 1 mM iodoacetate had no effect on the enzymatic rate, whereas 1 mM mercaptoethanol and maleic acid reduced the activity by only 5 to 10%. 10 mM p-Chloromercurybenzoate completely inactivates the enzyme, while 0.64 M beta-mercaptoethanol in the presence of 2 M urea causes only partial inactivation of the enzyme. 4 or 7 M Urea increases the enzyme activity.
  • Removal of Golden Nuclease
    BenzNuclease contain no "Tag" and used in downstream processing can be removed by various purification methods according to the purification strategy for the target protein.
  • Unit Definition
    One unit will digest sonicated salmon sperm DNA to acid-soluble oligonucleotides equivalent to a ΔA260 of 1.0 in 30 min at pH 8.0 at 37 ℃, which corresponds approximately to complete digestion of 37 μg DNA. Note that 1 KU=1000 units.
  • Formulation
    Lyophilized in Tris HCl, pH 8.0, MgCl2, and NaCl.
  • Concentration
    ≥250 U/μL
  • Reconstitution
    See Certificate of Analysis for reconstitution instructions and specific concentrations.
  • Storage
    No activity loss was observed after storage at:
                       In lyophilized state for 1 year (4oC); After reconstitution under sterile   conditions for 3 months (-70oC).
  • Purity
    >99% as determined by SDS-PAGE reduced BenzNuclease.
  • SDS-PAGE
    BenzNuclease

    Fig.1 The purity of BenzNuclease was determined by SDS-PAGE reduced and staining overnight with Coomassie Blue.

  • Activity Assay Procedure

    1. Reagents and solutions preparation

    Reaction buffer*:

    50 mM Tris-HCl, 1 mM MgCl2, pH 8.0 ( * In the case of extensive dilution before use, carrier protein such as 0.1 mg/ml HSA or BSA is generally recommended to avoid any enzyme loss from surface adsorption)

    DNA Substrate:

    1 mg/ml salmon sperm DNA is dissolved overnight at 4 ℃, in reaction buffer, and is then sonicated on ice to obtain a homogenous solution.

    Enzyme:

    Different dilution of nuclease with reaction buffer.

    Stop reagent:

    Trichloroacetic acid (TCA)

    2. Standard curve establishment

    400 μl substrate + 100 μl enzyme of known activity = 500 μl mixture

    1. Incubate the mixture at 37℃ for 30 min.

    2. Stop the reaction by addition of 400 μl cold TCA and incubate on ice for 10 min.

    3. Centrifuge at 8500g for 5 min.

    4. Measure the absorbance of supernatant at 260 nm.

    5. Lot a standard curve with nuclease of known activities for each set of measurements.

    3. Measurement of activity

    The activity of any unknown nuclease can be determined from a single measurement by means of the standard curve. The specific activity of BenzNuclease is >1.5 x 10e6 unit/mg protein.

  • Activity

    Fig 2. Standard curve of nuclease activity for BenzNuclease.

Please contact us via TechSupport@acrobiosystems.com if you have any question on this product.

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