Product Class: Other

Shrimp Alkaline Phosphatase (rSAP)
neb31 recombinant 37 65 Heat

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.

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
Note: See also Exo-CIP™ Rapid PCR Cleanup Kit (NEB #E1050).
Catalog # Size Concentration
M0371S 500.0 units 1000 units/ml
M0371L 2500.0 units 1000 units/ml

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    The Mechanism of Dephosphorylation

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).
This product is related to the following categories:
Phosphatases Products
This product can be used in the following applications:
Dephosphorylation,
RNA Cloning

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

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 rCutSmart™ Buffer
Incubate at 37°C

1X rCutSmart™ Buffer
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Storage Buffer

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

Heat Inactivation

65°C for 5 minutes

Molecular Weight

Theoretical: 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.

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.

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 (NEB #M0371)
  2. Enzymatic PCR Cleanup Protocol (NEB #M0371)

Usage & Guidelines

Application Notes

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Which alkaline phosphatase, Quick CIP, rSAP 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, Quick CIP, or Antarctic Phosphatase (AnP)?
  4. Does the DNA need to be purified after rSAP treatment?
  5. How stable is rSAP in its storage buffer at various temperatures?
  6. What is the effect of metal chelators, inorganic phosphate and phosphate analogs on rSAP activity?
  7. What is the effect of reducing agents on rSAP activity?
  8. Will Quick CIP, rSAP or Antarctic Phosphatase (AnP) dephosphorylate proteins?
  9. Can rSAP be heat inactivated?
  10. Does the DNA need to be purified after a restriction digest and prior to the dephosphorylation step?
  11. Does the DNA need to be purified after the dephosphorylation step and prior to the ligation step?
  12. Are the alkaline phosphatases active in NEBuffers?
  13. Cloning Problem:  Too much background in the transformation step.
  14. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?

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.

Specification Change Notifications

Effective 10/16/2023, buffer changed from CutSmart Buffer (NEB #B7204) to rCutSmart Buffer (NEB #B6004).

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

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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