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Shrimp Alkaline Phosphatase (SAP)

#EF0511 Supplied with: 10X Reaction Buffer	500 u (1 u/µl) 3x1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EF0511 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Active in Fermentas buffers for restriction enzymes, PCR and RT.
• Completely inactivative after 15 min incubation at 65°C.

Description
The Shrimp Alkaline Phosphatase (SAP) catalyzes the release of 5'- and 3'-phosphate groups from DNA, RNA and nucleotides. Also this enzyme can remove phosphate groups from proteins.

Source
Arctic shrimp Pandalus borealis.

Molecular Weight
This enzyme is homodimer. It consists of two identical subunits of 54 kDa (1).

Applications

• Dephosphorylation of DNA and RNA (2), see Protocol for Dephosphorylation of DNA 5'-termini and Protocol for DNA Digestion and Subsequent Dephosphorylation.
• Degradation of dNTPs in PCR mixture prior to sequencing of PCR products, see Protocol for PCR Product Clean-up (procedure according (3))
• Dephosphorylation of proteins (4), see recommendations below.

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested for dephosphorylation of DNA 5'-termini.

Concentration
1 u/µl

Definition of Activity Unit
One unit of the enzyme hydrolyzes 1 µmol of 4-nitrophenylphosphate in 1 min at 37°C.
Enzyme activity is assayed in the following mixture: 1 M diethanolamine-HCl (pH 9.8), 0.5 mM MgCl₂, 10 mM 4-nitrophenylphosphate.

Storage Buffer
The enzyme is supplied in: 25 mM Tris-HCl (pH 7.6 at 4°C), 1 mM MgCl₂, 0.1 mM ZnCl₂ and 50% (v/v) glycerol.

10X Reaction Buffer
0.1 M Tris-HCl (pH 7.5 at 37°C), 0.1M MgCl₂ and 1 mg/ml BSA.

Inhibition and Inactivation

• Inhibitors: metal chelators, inorganic phosphate and phosphate analogs.
• Inactivated by heating at 65°C for 15 min.

Note
The Shrimp Alkaline Phosphatase binding to DNA may result in a band shift in agarose gels. To avoid this, prior to electrophoresis we recommend to incubate the samples with Fermentas 6X Loading Dye & SDS Solution at 65°C for 10 min and chill on ice.

Recommendations for Dephosphorylation of Proteins

Reaction Mixture: 1X reaction buffer for SAP, 0.1-0.2 mg/ml of phosphoprotein, 10 u of Shrimp Alkaline Phosphatase. Incubate at 37°C for 1 hour. Note The reaction can be stopped by addition of EDTA to final 50 mM concentration or by addition of sodium orthovanadate (Na3VO4) to final 10 mM concentration. The optimal incubation time and the enzyme concentration must be determined experimentally for each substrate.

 

Related Products

T4 Polynucleotide Kinase Exonuclease I, E.coli T4 DNA Ligase T4 RNA Ligase Rapid DNA Ligation Kit Rapid DNA Ligation & Transformation Kit CloneJET™ PCR Cloning Kit 6X Loading Dye & SDS Solution Water, nuclease-free

References

1. Nilsen, I.W., et al., Thermolabile alkaline phosphatase from Northern shrimp (Padalus borealis): protein and cDNA sequence analyses, Comparative Biochemistry and Physiology, B, 129, 853-861, 2001.
2. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
3. Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, Nucleic Acids Res., 22, 4354-4355, 1994.
4. Khosravi, R., et al., Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage, Proc. Natl. Acad. Sci USA, 96, 14973-14977, 1999.

Updated vasario 25, 2008 10:23