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  • Pyrophosphatase, Inorganic (yeast)

    Description

    Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate.

    A variety of metabolic reactions generate inorganic pyrophosphate as a reaction byproduct. Such reactions are rendered irreversible when the pyrophosphate is degraded by pyrophosphatase (1). RNA and DNA synthesis are examples of reactions that can be pulled far in the synthesis direction by the action of inorganic pyrophosphatase.

    Highlights

    • Isolated from a recombinant source.

    Product Source

    PPase is prepared from an E. coli strain containing a genetic fusion of the Saccharomyces cerevisiae ppa gene and the gene coding for Mycobacterium xenopi GyrA intein. The final product has been cleaved and purified from the intein fusion partner. Developed by the Biohelix Corporation, and produced at New England Biolabs.

    Advantages and Features

    Applications

    • Enhancing yields of RNA in transcription reactions (2)

    Properties and Usage

    Unit Definition

    One unit is the amount of enzyme that will generate 1 µmol of phosphate per minute from inorganic pyrophosphate under standard reaction conditions (a 10 minute reaction at 25°C in 100 mM Tris-HCl, [pH 7.2], 2 mM MgCl2 and 2 mM PPi in a reaction volume of 0.5 ml).

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    100 mM KCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 8.0 @ 25°C

    Molecular Weight

    Theoretical: 71 kDa

    Quality Control

    Quality Assurance Statement

    • Free of endonuclease, exonuclease and RNase activities.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • dNTPase Activity:
      The Pyrophosphatase is tested in a reaction containing a mix of dNTP's. After incubation the amount of inorganic phosphatase released is determined by comparison to a standard curve of known phosphate concentrations.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    References

    1. Kornberg, A. (1980). DNA Replication. 54-55, 94. W.H. Freeman and Company..
    2. Cunningham, P.R. and Ofengand, J. (1990). Biotechniques. 9, 713-714.
    3. Cooperman, B.S. (1982). Methods Enzymol.. 87 (pt. C), 526-548.

    Supporting Documents

    Specifications

    The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Can alternate metals be used as activators for Yeast Inorganic Pyrophosphatase?