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Rapid Alkaline Phosphatase

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Rapid Alkaline Phosphatase For life science research only. Not for use in diagnostic procedures. rAPid Alkaline Phosphatase Orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1 Cat. No. 04 898 133 001 1000 units Cat. No. 04 898 141 001 5000 units y Version 03 Content version : May 2019 Store at Ϫ15 to Ϫ25°C 1. What this Product Does Enzyme Characteristics Number of Tests Parameter Description 1 kit is designed for Source Recombinant alkaline phosphatase from bovine intes- • 1000 dephosphorylation reactions (Cat. No. 04 898 133 001) tine (1) expressed in Pichia pastoris • 5000 dephosphorylation reactions (Cat. No. 04 898 141 001) Molecular 56 kD (by SDS-PAGE, monomer) with a final reaction volume of 20 ␮l each. weight 2+ Pack Contents Subunits homodimer (Zn is essential for activity) Unit Definition One unit of rAPid Alkaline Phosphatase is the enzyme Label Contents / Function activity which hydrolyzes 1 ␮mol of 4-nitrophenyl rAPid Alkaline Phos- 0.5 M Tris/HCl, 1mM EDTA, pH 8.5 (20°C) phosphatase in 1 min at 37°C under assay conditions. phatase Buffer, Volume 1 U/␮l 10ϫ conc. Activity rAPid Alkaline Phos- • 1000 U (Cat. No. 04 898 133 001) Specific Approx. 1U/␮g according to (2) and (3). phatase • 5000 U (Cat. No. 04 898 141 001) Activity See data label for lot-specific values. Storage and Stability Specificity Alkaline Phosphatase catalyzes the hydrolysis of numerous phosphate esters, such as esters of primary Ϫ Ϫ If stored at 15 to 25°C the product is stable through the expiration and secondary alcohols, saccharides, cyclic alcohols, date printed on the label. phenols and amines. Phosphodiesters do not react. The product is shipped on dry ice. The enzyme hydrolyzes inorganic pyrophosphate. The Storage buffer: 25 mM Tris/HCl; 1mM MgCl ; 1mM ZnCl ; 50 % kinetic properties of the enzyme depend on many fac- 2 2 tors, such as purity of enzyme, concentration of Glycerol (v/v); pH approx. 7.6 (4°C) enzyme in the assay, buffer, pH etc. Application Reaction 37°C rAPid Alkaline Phosphatase is used to dephosphorylate 5' ends of Temperature DNA and RNA. Alkaline phosphatase treatment prevents self-ligation Inactivation rAPid Alkaline Phosphatase is inactivated by incuba- of fragments by removing the 5'-phosphoryl termini required by tion at 75°C for 2 min. ligases; this feature is of major importance in cloning strategies to decrease vector background. rAPid Alkaline Phosphatase can also be Animal- none employed to dephosphorylate proteins. derived additives Use rAPid Alkaline Phosphatase in numerous applications, including: • Removal of 5'-phosphoryl groups from nucleic acids Assay Time • Preparation of templates for 5'-end labeling The assay time depends on the nature of the DNA ends to be dephos- • Clean-up of PCR products by removal of dNTPs phorylated: • Dephosphorylation of proteins •Up to 1 ␮g DNA with blunt or sticky 5' overhang ends: 10 min rAPid Alkaline Phosphatase is very rapidly inactivated by heating at dephosphorylation at 37°C 75°C for 2 min. No additional purification steps are required after •Up to 1 ␮g DNA with sticky 5' recessive ends: 30 min dephosphory- restriction and dephosphorylation. The dephosphorylated DNA can lation at 37°C directly be used in a ligation reaction. Dephosphorylation is followed by 2 min inactivation at 75°C. 0519.04951514001 sigma-aldrich.com 2. How to Use this Product Absence of nick- 1 ␮g"supercoiled" pBR322 DNA is incubated with ing activities various amounts of rAPid Alkaline Phosphatase Standard Dephosphorylation Procedure in 50 ␮l of all different SuRE/Cut Buffers available For standard dephosphorylation reactions, follow the procedure below. from Roche Diagnostics. This shows no decrease After restriction digestion with a Roche Diagnostics restriction or degradation of the supercoiled pBR322 DNA enzyme you can use the DNA directly in the dephosphorylation after agarose gel electrophoresis. step. No additional purification steps are required. References Step Action 1 Manes, T. et al. (1998). Genetic Complexity, Structure and Char- acterisation of Highly Active Bovine Intestinal Alkaline Phospha- ³ • Thaw all necessary components. tases. J. Biol. Chem. 273, 23353 – 23360. • Briefly centrifuge all reagents before starting. 2 Moessner, E. et al. (1980). Purification of human and bovine ᕢ • Add the following reagents to a reaction vial: alkaline phosphatase by affinity chromatography. Z. Physiol. Chem. 361, 543. Reagent Volume Final Con- centration 3 Bradford, M. (1976). A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the principle vector DNA x ␮lup to 1 ␮g of protein-dye binding. Anal. Biochem. 72, 248-254. rAPid Alkaline Phosphatase 2 ␮l1 ϫ ϫ Buffer, 10 conc. 4. Supplementary Information rAPid Alkaline Phosphatase 1 ␮l1U water, sterile deionized add to Ordering Information 20 ␮l Product Pack Size Cat No. Total Volume 20 ␮l Agarose MP ) 100 g 11 363 514 910 • Mix thoroughly and centrifuge briefly. 500 g ᕣ • Incubate DNA with blunt or sticky 5' overhang ends for T4 DNA Ligase 100 U [1 U/␮l] 10 481 220 001 10 min at 37°C. 500 U [1 U/␮l 10 716 359 001 500 U [5 U/␮l] 10 799 009 001 • Incubate DNA with sticky 5' recessive ends for 30 min at 37°C. T4 RNA Ligase 500 U 11 449 478 001 ᕤ Inactivate the rAPid Alkaline Phosphatase for 2 min at Agarose Gel DNA Extraction Kit 1 kit (max. 100 reac- 11 696 505 001 tions) 75°C. High Pure PCR Product Purifica- Kit for 50 purifications 11 732 668 001 ´ Either use the dephosphorylation reaction mixture tion Kit Kit for 250 purifications 11 732 676 001 directly in the following ligation reaction or store it at Ϫ Ϫ Water, PCR-grade 25 ml (25 vials of 1 ml) 03 315 932 001 15 to 25°C until further use. 25 ml (1 vials of 25 ml) 03 315 959 001 100 ml (4 vials of 03 315 843 001 25 ml) 3. Additional Information on this Product Changes to Pervious Version Product Description Editorial changes. rAPid Alkaline Phosphatase is isolated from bovine intestine and sup- plied as a recombinant enzyme expressed in the yeast, Pichia Pastoris. Trademarks The recombinant form ensures consistency and safety. rAPid Alkaline HIGH PURE and SURE/CUT are trademarks of Roche. Phosphatase catalyzes the dephosphorylation of 5' phosphates from All third party product names and trademarks are the property of DNA and RNA, nucleotides, and proteins. Unlike calf intestinal phos- their respective owners. phatase, rAPid Alkaline Phosphatase is rapidly, completely, and irre- versibly inactivated by heat treatment for two minutes at +75°C. It is Regulatory Disclaimer therefore an excellent alternative to Shrimp Alkaline Phosphatase. In For life science research only. Not for use in diagnostic procedures. addition, the enzyme is active in restriction enzyme buffers; therefore, restriction enzyme digestion, dephosphorylation, enzyme inactivation, Disclaimer of License ligation, or 5'-end labeling can be performed without purification For patent license limitations for individual products please refer to: steps. List of biochemical reagent products Background Information DNA ligases join linear DNA fragments together via covalent bonds. DNA ligation involves creating a phosphodiester bond between the 3' hydroxyl group of one nucleotide and the 5' phosphate of another. Ligation of DNA fragments is an essential step in many molecular biol- ogy techniques, including gene cloning and messenger RNA (mRNA) fingerprinting. For efficient ligation, DNA strands must be prevented Contact and Support from self-ligating (self-circularization and concatenation) by dephos- To ask questions, solve problems, suggest enhancements and report new phorylation of DNA ends. Alkaline phosphatase removes the 5'-phos- applications, please visit our Online Technical Support Site. phoryl termini required by ligases, preventing self-ligation of DNA and decreasing background. To call, write, fax, or email us, visit sigma-aldrich.com, and select your home country. Country-specific contact information will be displayed. Quality Control See data label for lot-specific values. Absence of 1 ␮g ␭ DNA is incubated with rAPid Alkaline deoxyribonucle- Phosphatase for 16 hrs in 50 ␮l of all different ases SuRE/Cut Buffers available from Roche Diagnos- tics. This shows no degradation of ␭ DNA after Roche Diagnostics GmbH Sandhofer Strasse 116 agarose gel electrophoresis. 68305 Mannheim Germany.
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