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Directions for Use Shrimp Alkaline Phosphatase, Recombinant (Rsap)

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Directions for Use Shrimp Alkaline Phosphatase, Recombinant (Rsap) Directions for Use Shrimp Alkaline Phosphatase, Recombinant (rSAP) Code Description Size 1B1633-1KU Shrimp Alkaline Phosphatase, Recombinant (rSAP) 1 vial, 1000 units (1 U/µL) 1B1633-5KU Shrimp Alkaline Phosphatase, Recombinant (rSAP) 5 vials, 1000 units each (1 U/µL) General Information Shrimp Alkaline Phosphatase, Recombinant (rSAP) is a heat-labile hydrolase enzyme produced in Pichia Pastoris that removes phosphate groups nonspecifically from 5’ ends of nucleic acid phosphomonoesters and proteins. This activity is most commonly utilized in molecular cloning to prevent self-ligation of linearized plasmid DNA and in 5’ end-labeling to facilitate the replacement of unlabeled phosphates with labeled phosphate groups. rSAP also prepares PCR products for DNA sequencing or SNP analysis, by dephosphorylating unincorporated dNTPs that would otherwise interfere with enzymatic reactions. VWR Life Science AMRESCO’s rSAP may be directly added to restriction enzyme digests and is conveniently inactivated 100% by heating at 65°C. This eliminates the need for vector purification, a step that is necessary when using alkaline phosphatases isolated from other sources, such as E. coli and calf intestine. rSAP works well in common buffers and does not require supplemental zinc or other additives. Removes 5’-phosphates from DNA, RNA, dNTPs and proteins Improves cloning efficiency by preventing vector recircularization 100% heat-inactivated at 65°C No vector purification necessary Removes unincorporated dNTPs in PCR products prior to DNA sequencing or SNP analysis Prepares templates for 5’-end labeling Dephosphorylates proteins Works in many different buffers without supplemental factors AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use Storage/Stability Stable at least 2 years when stored frozen (0 to -20°C). When stored cold (2 – 8°C), rSAP is stable for 6 months. At room temperature (18 – 26°C), rSAP is stable for 3 months. rSAP tolerates multiple freeze-thaw cycles. Product Use Limitations For research use only. Not for therapeutic or diagnostic use. Protocol/Procedure: Notes: One unit of rSAP releases 1 µmol phosphate/minute from 4-nitrophenyl phosphate in 0.1 M glycine-NaOH pH 10.4, 1 mM MgCl2, 1 mM ZnCl2 and 6 mM 4-nitrophenyl phosphate. The resulting p-nitrophenol generated is measured by absorption at 405 nm. rSAP works well in most restriction digestion and PCR reaction buffers within the optimum pH range of 7 – 9. Activity requires > 1 mM Mg2+. Complete inactivation of rSAP is achieved by incubation at 65°C for 5 minutes. Dephosphosphorylation with rSAP During Restriction Digestion Reaction 1. Digest 1 µg of plasmid DNA in a 50 µL reaction according to the table below. Note: Use 0.1 U rSAP per U of restriction enzyme. Restriction cutting reaction may vary from example below. Follow the supplier’s protocol for the restriction enzyme to be used. Reaction Component Volume 1 µg Plasmid DNA >1 µL 10X restriction enzyme buffer 5 µL 10 U Restriction enzyme 0.5 µL 1 U shrimp alkaline phosphatase 1 µL recombinant Water Bring final volume to 50 µL 2. Incubate the reaction at 37°C for 1 hour. 3. Heat 5 minutes at 65°C to inactivate Shrimp Alkaline Phosphatase Recombinant, or follow heat inactivation protocol recommended for the restriction enzymes in use. 4. Proceed to ligation protocol. AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use Dephosphosphorylation with rSAP After Restriction Digestion Reaction 1. Digest plasmid DNA using the supplier’s protocol for the restriction enzyme to be used. 2. After digestion, add 5 U Shrimp Alkaline Phosphatase, Recombinant per µg of plasmid to the reaction mixture. 3. Incubate at 37°C for an additional 10 minutes. 4. Proceed to ligation protocol. Clean-up of PCR Products Note: In this protocol, rSAP dephosphorylates nucleotides and Exonuclease I degrades primers to prepare PCR products for sequencing or genotyping. 1. After performing PCR, add 2 U rSAP and 10 U Exonuclease I to the reaction tube. 2. Incubate at 37°C for 15 minutes. 3. Heat inactivate at 80°C for 15 minutes. 4. Proceed to sequencing or genotyping protocol, or store product at -20°C until required Frequently Asked Questions What is the source of AMRESCO’s rSAP? The recombinant SAP is produced in Pichia pastoris and is designed to replace the native enzyme originating in arctic shrimp. How does the activity of Shrimp Alkaline Phosphatase, Recombinant (rSAP) compare to the native enzyme? rSAP functions as well as native SAP and may be implemented directly without any changes to protocols. What is the stability of rSAP? Compared to native SAP, rSAP is significantly more stable at ambient temperature, with > 95% of rSAP activity retained after 90 days of room temperature storage. How is a Unit of rSAP defined? One Unit of rSAP converts 1 µmol of p-nitrophenyl phosphate per minute to nitrophenol and phosphate at 37°C and pH 10.4 in 0.1 M glycine buffer containing 1 mM each of ZnCl2, MgCl2 and 6 mM 4-nitrophenyl phosphate. How does the activity of AMRESCO’s rSAP compare to a similar competing product? Based on the Unit definition, 1 Unit of rSAP is equivalent to 5 – 40 Units of Antarctic Phosphatase (New England BioLabs). AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use How should rSAP be heat inactivated? rSAP is inactivated after incubation for 5 minutes at 65°C. Complete inactivation is achieved after only 1 minute of incubation at 75°C. In a standard thermocycler, the process of heating from 37 – 95°C and dropping back to 37°C is sufficient to completely inactivate rSAP. Which phosphate groups are removed by rSAP? 5’- phosphates from single- and double stranded DNA and RNA, as well as 3’- and 2’- monphosphates from DNA are removed by rSAP. A literature search also reveals applications for rSAP in protein dephosphorylation4,5. Are protruding, recessed and blunt 5’- double-stranded DNA ends efficiently dephosphorylated by rSAP? Yes, rSAP dephosphorylates protruding, recessed and blunt 5’- ends efficiently. Following rSAP treatment of linearized vector plasmid, should the DNA be purified prior to ligation? Vector DNA treated with rSAP does not need to be purified prior to ligation as long as complete inactivation of rSAP is achieved by heating at least 5 minutes at 65°C. Does rSAP withstand freeze-thaw cycles? Yes, rSAP retains high activity after multiple freeze-thaw cycles when stored in its original storage buffer. Are there any additives that should be added to reactions for rSAP to function? The rSAP storage buffer supplies the Zn2+ amd Mg2+ required for its activity. rSAP performs very well in most common reaction buffers without addition of any further additives. Does AMRESCO provide a protocol for dephosphorylate of proteins using rSAP? AMRESCO does not provide a recommended protocol for protein dephosphorylation using rSAP. A review of scientific literature will yield publications with protocols that may be followed4,5. References 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. AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 Directions for Use 3. 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. 4. 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. 5. 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. 6. Method enabling pyrosequencing on double-stranded DNA. Nordström T., Nourizad K., Ronaghi M., Nyrén P. (2000) Anal Biochem. 282(2): 186-93. 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. For Technical Support Toll Free: 1-800-610-2789 (USA & Canada) Fax: (440) 349-0235 Email: [email protected] AMRESCO, LLC A VWR Company Corporate Headquarters 28600 Fountain Parkway Solon, Ohio USA 44139-4300 Tel: 440/349-1199 Fax: 440/349-1182 www.amresco-inc.com Shrimp Alkaline Phosphatase, Recombinant (rSAP) ZY0601 Rev. 1 12/2015 © Copyright 2010 by AMRESCO, LLC All Rights Reserved. AMRESCO® is a registered trademark of AMRESCO, LLC AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139 .
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