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  • Shrimp Alkaline Phosphatase (rSAP)

    Description

    Shrimp Alkaline Phosphatase (rSAP) is a heat labile alkaline phosphatase purified from a recombinant source. rSAP is identical to the native enzyme and contains no affinity tags or other modifications. rSAP nonspecifically catalyzes the dephosphorylation of 5´ and 3´ ends of DNA and RNA phosphomonoesters. Also, rSAP hydrolyses ribo-, as well as deoxyribonucleoside triphosphates (NTPs and dNTPs). rSAP is useful in many molecular biology applications such as the removal of phosphorylated ends of DNA and RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents religation of linearized plasmid DNA. The enzyme acts on 5´ protruding, 5´ recessed and blunt ends. rSAP may also be used to degrade unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis. rSAP is completely and irreversibly inactivated by heating at 65°C for 5 minutes, thereby making removal of rSAP prior to ligation or end-labeling unnecessary.

    Product Source

    A Pichia pastoris clone that carries the shrimp alkaline phosphatase gene from Northern shrimp Pandalus borealis (1,2).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    CutSmart™ Buffer-2010X

    Advantages and Features

    Features

    • Rapid and irreversible heat inactivation eliminates unwanted activity
    • Improved storage stability versus native enzyme
    • Faster reaction setup (no supplemental additives like zinc required)
    • Flexible reaction conditions (active in any restriction enzyme buffer, no clean-up required)
    • Less enzyme required (high specific activity), resulting in a lower cost per reaction
    • No need for multiple phosphatases (rSAP removes 5´- and 3´- phosphates from DNA, RNA and dNTPs )
    • Active on unincorporated dNTPs in PCR products - improves DNA sequencing and SNP analysis
    • Recombinant for purity, consistency and value

    Figure 1: rSAP heat inactivation at 65°C.

    1 unit of rSAP was incubated under recommended reaction conditions, including DNA, for 30
    minutes and then heated at 65°C. Remaining phosphatase activity was measured by PNPP assay.

    Applications

    • Dephosphorylation of cloning vector DNA to prevent recircularization during ligation.
    • Dephosphorylation of DNA prior to end-labeling using T4 Polynucleotide Kinase.
    • Treatment of dNTPs in PCR reactions prior to sequencing or SNP analysis.
    • Dephosphorylation of DNA and RNA.

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that hydrolyzes 1 μmol of p-Nitrophenyl Phosphate, PNPP (NEB #P0757) in a total reaction volume of 1 ml in 1 minute at 37°C.

    Reaction Conditions

    1X CutSmart™ Buffer
    Incubate at 37°C

    1X CutSmart™ Buffer:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    100 μg/ml BSA
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    25 mM Tris-HCl
    1 mM MgCl2
    50% Glycerol
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 5 min

    Molecular Weight

    Calculated: 54 kDa1

    Specific Activity

    3,000 units/mg

    Unit Assay Conditions

    1 M diethanolamine-HCl (pH 9.8), 0.5 mM MgCl2, 50 mM p-Nitrophenyl Phosphate. These conditions are only used for quantitating enzyme activity.

    Quality Control

    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.
    • 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.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • RNase Activity (Extended Digestion):
      The product is tested in a reaction containing a RNA substrate. After incubation for 16 hours greater than 90% of the substrate RNA remains intact as determined by gel electrophoresis.

    Notes

    1. Molecular Weight: rSAP is a homodimer. The molecular weight of the monomer is 54 kDa.
    2. rSAP, as are most alkaline phosphatases, is a Zn2+ and Mg2+-dependent enzyme. Our formulation has tightly bound zinc atoms in the active center and does not require supplemental zinc or other additives.
    3. rSAP is also active in 1X NEBuffers 1.1, 2.1, 3.1 as well as NEBuffers 1, 2, 3, 4 and NEBuffer for EcoRI.
    4. rSAP activity is enhanced in the presence of monovalent salts.
    5. rSAP is inhibited by metal chelators (e.g. EDTA), inorganic phosphate and phosphate analogs.
    6. The rSAP activity is decreased in the presence of reducing agents (DTT, β-ME).

    References

    1. Olsen, R. L. et al. (1991). Comp. Biochem. Physiol. 99B(4):, 755-761.
    2. Nilsen, I.W. et al. (2001). Comp. Biochem. Physiol. 129B(4):, 853-861.

    Supporting Documents

    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. Which alkaline phosphatase, CIP, Antarctic or rSAP, works best?
    2. The number of colonies that do not contain an insert seems high. How can I tell if rSAP worked?
    3. What phosphate groups are removed by rSAP (or CIP or AnP)?
    4. Will rSAP work in restriction enzyme NEBuffers?
    5. Does the DNA need to be purified after rSAP treatment?
    6. How stable is rSAP in its storage buffer at various temperatures?
    7. What is the effect of metal chelators, inorganic phosphate and phosphate analogs on rSAP activity?
    8. What is the effect of monovalent salts on rSAP activity?
    9. What is the effect of reducing agents on rSAP activity?
    10. Will rSAP (or CIP or AnP) dephosphorylate proteins?
    1. Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371)
    2. Protocol for Dephosphorylation of 5´-ends of DNA using rSAP in Restriction Enzyme Reaction (M0371)

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