Shrimp Alkaline Phosphatase

Overview

Shrimp Alkaline Phosphatase (SAP) – still the golden standard and the first heat-labile alkaline phosphatase on the market. It is a multipurpose alkaline phosphatase that can be fully inactivated by a short heat treatment.

SAP has been on the market since 1993 and is now replaced by the new and better recombinant form. The Recombinant enzyme is produced in Pichia Pastoris, and has all the properties of the well-proven native version, but with additional benefits. The recombinant version is far more stable at ambient temperature, is also of high, consistent purity, and available at larger batches at high concentration.

The main advantages of SAP

  • 100% heat-inactivated at 65°C
  • Removes 5’-phosphates from DNA, RNA, dNTPs and proteins
  • Works in restriction enzyme buffers
  • Used in PCR clean-up

Properties

Specifications

Unit definition: One Unit will convert 1 µmol of p-nitrophenyl phosphate per minute to nitrophenol and phosphate at 37ºC and pH 10.4 in 0.1 M glycine buffer, 1 mM each of ZnCl2 and MgCland 6 mM 4-nitrophenyl phosphate.

This Unit definition makes 1 Unit of SAP equivalent to 5 to 40 Units of Antarctic Phosphatase (New England Biolabs), 1.3 Units APex phosphatase or 35 Units NTPhos phosphatase (Epicentre Biotechnologies). SAP is therefore the most processive alternative.

  • Specific activity: >2 000 Units/mg.
  • Purity: Purified to apparent homogeneity by SDS-PAGE. DNase, RNase or protease activities not detected.
  • Concentration: Minimum 10 000 Units/ml, available up to 30 000 Units/ml.
  • Batch sizes: 25 – 35 million Units / batch.

Stability

Stable at -20ºC in storage buffer (25 mM Tris-HCl pH 7.6, 5 mM MgCl2, glycerol 50%). At room temperature, >95% activity remains after 90 days.

Still, the enzyme is completely inactivated after 5 minutes at 65°C. At 75°C, SAP is completely inactivated after only 1 minute. In a standard thermocycler, the process of heating from 37 to 95 and back to 37°C is sufficient to completely inactivate SAP.

Intellectual property

Recombinant SAP is covered by the following patents: US 7,323,325, EP 1,326,890, JP 4,191,479, AU 9,398,701 and related applications in other countries either granted or pending.

Publications

Descriptive papers

  1. Ligand-binding and metal-exchange crystallographic studies on shrimp alkaline phosphatase.
    de Backer M.M., McSweeney S., Lindley P.F., Hough E. (2004)
    Acta Crystallogr D Biol Crystallogr. 60(Pt 9):1555-61. Epub.  Aug 26. 2004
  2. Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses.
    Nilsen I.W., Øverbø K., Olsen R.L. (2001)
    Comp Biochem Physiol B Biochem Mol Biol. 129(4): 853-61.

Papers showing use of SAP

Dephosphorylation of cloning vectors

  1. Generating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination
    Jeongmin Lee, Eugene Rha, Soo-Jin Yeom, Dae-Hee Lee, Eui-Sung Choi and Seung-Goo Lee* (2013)
    PLoS One. 2013; 8(11): e79979. doi: 10.1371/journal.pone.0079979

Degradering of nucleotides before genotyping or sequencing

  1. Medium-throughput SNP genotyping using mass spectrometry: multiplex SNP genotyping using the iPLEX® Gold assay.
    Millis M.P. (2011)
    Methods Mol Biol. 700:61-76.
  2. Highly multiplexed genotyping of thiopurine s-methyltransferase variants using MALD-TOF mass spectrometry: reliable genotyping in different ethnic groups.
    Schaeffeler E., Zanger U.M., Eichelbaum M., Asante-Poku S., Shin J.G., Schwab M. (2008)
    Clin Chem. 54(10): 1637-47. Epub.  Aug 7. 2008.
  3. Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices.
    Ahlford A., Kjeldsen B., Reimers J., Lundmark A., Romani M., Wolff A., Syvänen A.C., Brivio M. (2010)
    Analyst. 135(9): 2377-85. Epub Jul 29. 2010.
  4. Genotyping SNPs using a UV-photocleavable oligonucleotide in MALDI-TOF MS.
    Vallone P.M., Fahr K., Kostrzewa M. (2005)
    Methods Mol Biol. 297: 169-78.
  5. A gel-free SNP genotyping method: bioluminometric assay coupled with modified primer extension reactions (BAMPER) directly from double-stranded PCR products.
    Zhou G.H., Shirakura H., Kamahori M., Okano K., Nagai K., Kambara H. (2004)
    Hum Mutat. ug. 24(2): 155-63.
  6. G2 checkpoint in uterine cervical cancer with HPV 16 E6 according to p53 polymorphism and its screening value.
    Cho N.H., Lim S.Y., Kim Y.T., Kim D., Kim Y.S., Kim J.W. (2003)
    Gynecol Oncol. 90(1): 15-22.
  7. A novel procedure for efficient genotyping of single nucleotide polymorphisms.
    Sauer S., Lechner D., Berlin K., Lehrach H., Escary J.L., Fox N., Gut I.G. (2000)
    Nucleic Acids Res. 28(5): E13.
  8. ExoSAP-IT™
    Affymetrix (previously USB) has for several years utilized SAP together with Exonuclease I (ExoSAP-ITTM), to offer a simple, fast and cost efficient way to purify PCR products before Sequencing and genotyping. When PCR amplification is complete, any dNTPs or primers remaining in the PCR mixture will interfere with downstream applications. ExoSAP-It removes these contaminants.

Protein dephosforylation

  1. Autophosphorylation activates Dictyostelium myosin II heavy chain kinase A by providing a ligand for an allosteric binding site in the alpha-kinase domain.
    Crawley S.W., Gharaei M.S., Ye Q., Yang Y., Raveh B., London N., Schueler-Furman O., Jia Z., Côté G.P. (2011)
    J Biol Chem. 286(4): 2607-16. Epub Nov 11. 2010.
  2. A diurnally regulated dehydrin from Avicennia marina that shows nucleo-cytoplasmic localization and is phosphorylated by Casein kinase II in vitro.
    Mehta P.A., Rebala K.C., Venkataraman G., Parida A. (2009)
    Plant Physiol Biochem. 47(8): 701-9. Epub Mar 28. 2009.

SAP for quantification

  1. Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters.
    Wadler C., Cronan J.E. (2007)
    Anal Biochem. 368(1): 17-23. Epub Jun 7. 2007.
  2. A comparison of enzymatic digestion for the quantitation of an oligonucleotide by liquid chromatography-isotope dilution mass spectrometry.
    Donald C.E., Stokes P., O’Connor G., Woolford A.J. (2005)
    J Chromatogr B Analyt Technol Biomed Life Sci. 817(2): 173-82.

For pyrosequensing

  1. Method enabling pyrosequencing on double-stranded DNA.
    Nordström T., Nourizad K., Ronaghi M., Nyrén P. (2000)
    Anal Biochem. 282(2): 186-93.