Phosphatase, Alkaline, Shrimp, Recombinant, BioAssay(TM)

Recombinant Shrimp Alkaline Phosphatase (rSAP) is a high specific activity, heat-labile alkaline phosphatase purified from a recombinant source and originally isolated from Pandalus borealis (arctic shrimp). rSAP 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. rSAP may also be used to treat unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis. \nrSAP 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, rSAP is completely and irreversibly inactivated by heating reactions at 65 degrees C for 15 min.\nrSAP 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. rSAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step.\n\nKit Components:\nP4071-05B1: Phosphatase, Alkaline, Shrimp, Recombinant (rSAP) (1unit/ul), 1x500u\nP4071-05B2: Shrimp Alkaline Phosphatase (SAP) Reaction Buffer (10X), 1x1ml\nP4071-05B3: Shrimp Alkaline Phosphatase Dilution Buffer, 1x500ul\n\nrShrimp 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. rSAP dephosphorylates all of the remaining dNTPs from the PCR mixture in one easy step.\nThe enzymatic activity and heat inactivation profile of recombinant is identical to nSAP.\n\nпїЅпїЅ100% heat-inactivated in 5 min at 65 degrees C\nпїЅпїЅSignificantly improved storage stability at lower temperatures\nпїЅпїЅVery high specific activity \nпїЅпїЅRemoves 5пїЅ-phosphates from DNA, RNA, dNTPs and proteins \nпїЅпїЅMay be added directly to restriction enzyme digests \nпїЅпїЅNo vector purification necessary \nпїЅпїЅRequires no supplemental zinc or other additives for activity \nпїЅпїЅWorks direct in many different buffers \nпїЅпїЅEasy treatment of unincorporated dNTPs in PCR products prior to DNA sequencing or SNP analysis\n\nMolecular Weight: Homodimer. Monomer is 55kD as determined by amino acid sequence.\nOptimum pH: 10.4 in glycine buffer and pH 8.0 in Tris buffer.\nOptimum Temperature: 37 degrees C\nHeat-Inactivation: 65 degrees C for 15 min.\nInhibitors: 10mM DTT, 0.1% b-ME\nReaction Conditions: \nActive in sodium chloride, potassium chloride. Requires Mg2+ for highest activity.\n\nPurity:\nTested for contaminating endonucleases, exonucleases and ribonucleases.\n\nStorage Buffer:\n25mM Tris-HCl, pH 7.5, 1mM MgCl2, 50% glycerol.\n\nAssay Conditions:\nThe reaction mixture contains 100mM glycine, pH 10.4, 1mM magnesium chloride, 10mM p-nitrophenyl phosphate and 0.001-0.1 units of P4071-05B1, recombinant Shrimp Alkaline Phosphatase (rSAP). The change in absorbance at 405nm is monitored (3050ul reaction volume).\n\nUnit Definition:\nOne unit is the amount of enzyme which catalyzes the hydrolysis of 1umol of p-nitrophenyl phosphate per min in glycine buffer, pH 10.4 at 37 degrees C.\n\nConcentration:\n1 unit/ul\n\nFunctional Assay:\nDephosphorylation of restriction enzyme digested plasmids (5-20 pmol of 5'-ends, 0.1-0.5units/pmol 5'-ends). Reduces religation and transformation to < 0.5% compared to an untreated control.\n\nStorage and Stability:\nFor long-term storage, aliquot and store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

P4071-05B

Size

100U

References

Recombinant references: 1. Werle, E., Schneider C., Renner, M., Volker, M. and Fiehn, W. (1994) Nucleic Acids Res. 22, 4354-4355. 2. Hanke, M. and Wink, M. (1994) BioTechniques 17, 858-860. (Native): 1. Haller, G. et al., (2009) J. Allergy and Clinical Immunology 124:1204-1209. 2. Rudge, S.A. et al., (1998) J. Cell. Biol. 140:181-90. General References: 1. Ruan, C. C., Samols, S. B., Fuller, C. W. (1990) Comments 17, (No.1), United States Biochemical Corporation, Cleveland, OH. 2. Werle, E., Scneider C., Renner, M., Volker, M., Fiehn, W. (1994) Nucleic Acids Res. 22, 4354-4355. 3. Hanke, M., Wink, M. (1994) BioTechniques 17, 858-860.