Shrimp Alkaline Phosphatase (SAP)
High specific activity, heat-labile alkaline phosphatase purified from a recombinant source
Proven Performance – the Phosphatase benchmark
- 100% heat-inactivated in 5 min at 65°C (see Fig. 1)
- Significantly improved storage stability at lower temperatures (see Fig. 2)
- Very high specific activity (see Fig. 3)
- Removes 5’-phosphates from DNA, RNA, dNTPs and proteins
- Purified from a recombinant source
- May be added directly to restriction enzyme digests
- No vector purification necessary
- Requires no supplemental zinc or other additives for activity
- Works direct in many different buffers
- Easy treatment of unincorporated dNTPs in PCR products prior to DNA sequencing or SNP analysis
USB Shrimp Alkaline Phosphatase (SAP)
Shrimp Alkaline Phosphatase (SAP) is a high specific activity, heat-labile alkaline phosphatase purified from a recombinant source and originally isolated from Pandalus borealis (arctic shrimp). SAP is useful in many molecular biology applications such as the dephosphorylation of phosphorylated ends of DNA or RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents relegation of linearized plasmid DNA. SAP may also be used to treat unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.
Shrimp Alkaline Phosphatase has approximately the same specific activity as Calf Intestinal Alkaline Phosphatase (CIAP), and like CIAP, is active in virtually all restriction enzyme reaction buffers. Unlike CIAP, Shrimp Alkaline Phosphatase is completely and irreversibly inactivated by heating reactions at 65°C for 15 min.
Shrimp Alkaline Phosphatase is particularly useful in preparing PCR products for applications involving sequencing, SNP analysis or labeling methods. Typically, excess dNTPs remaining after PCR interfere with subsequent enzymatic reactions involving DNA synthesis. SAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step.
Affymetrix is the world-wide provider of USB Shrimp Alkaline Phosphatase and we are pleased to be offering a recombinant version of our phosphatase benchmark. Recombinant SAP eliminates the dependence on animal sourcing and offers the added benefits of increased storage stability and batch to batch consistency while providing exceptional enzymatic activity and 100% heat inactivation.
Molecular Weight: Homodimer. Monomer is 55 kDa as determined by amino acid sequence.
Optimum pH: 10.4 in glycine buffer and pH 8.0 in Tris buffer.
Optimum Temperature: 37°C
Heat-Inactivation: 65°C for 15 min.
Inhibitors: 10mM DTT, 0.1% β-ME
Reaction Conditions: Active in NaCl, KCl. Requires Mg2+ for highest activity.
Tested for contaminating endonucleases, exonucleases and ribonucleases.
25mM Tris-HCl (pH 7.5), 1mM MgCl2, 50% glycerol.
The reaction mixture contains 100mM glycine, pH 10.4, 1mM MgCl2, 1mM ZnCl2, 10mM p-nitrophenyl phosphate and 0.001-0.1 units of recombinant Shrimp Alkaline Phosphatase (rSAP). The change in absorbance at 405 nm is monitored (3050 μl reaction volume).
One unit is the amount of enzyme which catalyzes the hydrolysis of 1 μmol of p-nitrophenyl phosphate per min in glycine buffer (pH 10.4) at 37°C.
Tested User Friendly™ Functional Test:
Dephosphorylation of restriction enzyme digested plasmids (5 – 20 pmol of 5'-ends, 0.1 -0.5 units/pmol 5'-ends). Reduces religation to < 0.5% compared to the untreated control.
PROTOCOL FOR DEPHOSPHORYLATION OF NUCLEOTIDES AND DEGRADATION OF PRIMERS PRIOR TO SEQUENCING REACTIONS OR SNP ANALYSES:
Please refer to the USB ExoSAP-IT protocol, the benchmark in PCR clean-up.
The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.
Functionally Tested 10X SAP Reaction Buffer (Included, PN 70103):
200mM Tris-HCl (pH 8.0), 100mM MgCl2.
Functionally Tested SAP Dilution Buffer (1 ml included, PN 72761):
50mM Tris-HCl (pH 8.0).
RUAN, C. C., SAMOLS, S. B. AND FULLER, C. W. (1990) Comments 17, (No.1), United States Biochemical Corporation, Cleveland, OH.
WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
HANKE, M. AND WINK, M. (1994) BioTechniques 17, 858-860.
- Dephosphorylation of DNA prior to cloning.
- PCR Clean-Up for sequencing and SNP applications.
- Dephosphorylation of DNA prior to end-labelling using T4 PNK or OptiKinase
- Dephosphorylation of RNA.
- Protein dephosphorylation.
Shipped on dry ice. Store at -20°C.