Product Class: Other

Shrimp Alkaline Phosphatase (rSAP)
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Item# Description List Price Web Price Qty
M0371L Shrimp Alkaline Phosphatase (rSAP) - 2,500 units $347.00
$312.30
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M0371S Shrimp Alkaline Phosphatase (rSAP) - 500 units $86.00
$77.40
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

New! Quick Dephosphorylation Kit offers heat inactivation, high specific activity and a fast protocol.

    Product Introduction

    Shrimp Alkaline Phosphatase (rSAP) is a heat labile alkaline phosphatase purified from a recombinant source.

    • 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
     

    Product Information

    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.

    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.

    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 -20 10X
    Product Categories:
    Phosphatases & Sulfurylases Products,
    RNA Modification Products
    Applications:
    Dephosphorylation,
    RNA Modification,
    In vitro Synthesis (IVT)

    Advantages and Features

    Application Features

    • Dephosphorylation 5´ and 3´ ends of DNA and RNA.
    • Dephosphorylation of cloning vector DNA to prevent recircularization during ligation.
    • Dephosphorylation of DNA prior to end-labeling using T4 Polynucleotide Kinase.
    • Removal of dNTPs in PCR reactions prior to sequencing or SNP analysis.

    Properties & 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 kDa

      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.

      Product Notes

      1. 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.
      2. 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.
      3. rSAP activity is enhanced in the presence of monovalent salts.
      4. rSAP is inhibited by metal chelators (e.g. EDTA), inorganic phosphate and phosphate analogs.
      5. The rSAP activity is decreased in the presence of reducing agents (DTT, β-ME).
      6. Molecular Weight: rSAP is a homodimer. Themolecular weight of the monomer is 54 kDa.

      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.

      Protocols, Manuals & Usage

      Protocols

      1. Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371)
      2. Enzymatic PCR Cleanup Protocol

      Usage & Guidelines

      • Activity of DNA Modifying Enzymes in CutSmart® Buffer

      Application Notes

      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 [email protected] or fill out the Technical Support Form for appropriate document.)

      Tools & Resources

      Selection Charts

      • Buffer and Diluent Formulation Table
      • Phosphatase Selection Chart

      Web Tools

      Faqs & Troubleshooting

      FAQs

      1. Which alkaline phosphatase, Quick CIP, from the Quick Dephosphorylation Kit, rSAP, CIP or Antarctic Phosphatase 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, CIP, Quick CIP, or AnP?
      4. Will rSAP work in restriction enzyme NEBuffers, including CutSmart?
      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 reducing agents on rSAP activity?
      9. Will Quick CIP, rSAP, CIP, or AnP dephosphorylate proteins?
      10. Can rSAP be heat inactivated?
      11. Does the DNA need to be purified after a restriction digest and prior to the dephosphorylation step?
      12. Does the DNA need to be purified after the dephosphorylation step and prior to the ligation step?
      13. Are the alkaline phosphatases active in NEBuffers?
      14. Cloning Problem:  Too much background in the transformation step.
      15. Which alkaline phosphatase, rSAP, CIP, or Antarctic Phosphatase, works best?
      16. Which alkaline phosphatase, rSAP, CIP, or Antarctic Phosphatase, works best?

      Troubleshooting

      Quality, Safety & Legal

      Quality Assurance Statement

      Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

      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]

      Certificate Of Analysis

      The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

      Safety DataSheets

      The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

      Legal and Disclaimers

      This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

      While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

      For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

      This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.