Antarctic Phosphatase

Item Number

A743821

• Specifications & Purity: free of DNA exonuclease and endonuclease, free of RNAase.
• Stability And Storage: Store at -20℃.
• Storage Temp: Store at -20°C
• Shipped In: Ice chest + Ice pads

Product Description

Aladdin's Antarctic Phosphatase (Thermosensitive) is a recombinant thermosensitive phosphatase that catalyzes the dephosphorylation of DNA, RNA, dNTP, and NTP. It can also be used to remove phosphate groups from serine, threonine or tyrosine residues of protein. Dephosphorylation of 5' and 3' ends of DNA prevents religation of linearized plasmid in cloning, which can significantly increase positive clones.Antarctic Phosphatase (AnP) is a new type of alkaline phosphatase that can maintain 100% activity in various endonuclease and PCR buffers of aladdin. It can accomplish the dephosphorylation of various types of DNA by incubating at 37℃ for 10 minutes, and can be completely inactivated by incubating at 75℃ for 5 minutes.Alkaline Phosphatase (AP/ALP/AKP/ALKP/ALPase/Alk Phos) is a class of hydrolases and most effective under alkaline conditions. Ir removes phosphate groups from nucleic acids, nucleotides, proteins and alkaloids by hydrolyzing phosphate monoesters and generating phosphate ions and free hydroxyl groups.Main features of this product

Application：

prevention of relignation of vectors or DNA fragments by removing phosphate groups at 5' ends; simultaneous endonuclease digestion and dephosphorylation of plasmid DNA; preparation of labeled DNA or RNA at 5' ends with T4PNK; removal of 5'-phosphate groups of DNA, RNA, NTP and dNTP; removal of dNTP and pyrophosphate from PCR products; dephosphorylation of serine, threonine and tyrosine residues of proteins.

Source：

Antarctic Phosphatase recombinantly expressed in E. coli. Antarctic Phosphatase typically forms homodimers with a molecular weight of 70kD.

Enzyme storage buffer：

10mM Tris-HCl (pH 7.4), 1mM MgCl2, 0.1mM ZnCl2, 50% (v/v) glycerol.

Inactivation or inhibition：

Antarctic Phosphatase can be completely inactivated by heating at 75℃ for 5 minutes. Metal chelators such as EDTA can inhibit Antarctic Phosphatase activity.Activity assay of Antarctic Phosphatase before and after inactivation at 75℃.Figure 1. Activity assay of Antarctic Phosphatase before (triangles) and after (circles) inactivation at 75℃. The colorimetric assay was performed with pNPP substrate and terminated by heating at 75℃ for 5 min. Reactions of 50µl containing 10mM pNPP and 2µl of 125-fold diluted Antarctic Phosphatase were incubated at room temperature for 20 minutes and the absorbance at 405nm of each reaction was measured kinetically for every one minute.This product can greatly reduce the religation of linearized vector DNA `by single restriction enzyme (Figure 2).Figure 2. Dephosphorylation of BamHI-digested pUC18 vector by Antarctic Phosphatase can greatly reduce recircularization of pUC18. A. Ligation of linearized pUC18 vector without dephosphorylation by Antarctic Phosphatase, and the ligation product was transformed into DH5α; B. Ligation of linearized pUC18 vector after dephosphorylation by Antarctic Phosphatase, and the ligation product was transformed into DH5α.Dephosphorylation of double digested vectors with this product prior to ligation can greatly increase the positive rate of clones (Figure 3).Figure 3. Antarctic Phosphatase greatly increased positive clones by dephosphorylation of pUC18 vector digested with EcoRI and BamHI. A and C. Clones (A) and Colony PCR results (C) obtained from transformation of DH5α cells with the ligation product of 600bp PCR products with the EcoRI/BamHI-digested pUC18 without dephosphorylation; B and D. Clones (B) and Colony PCR results (D) obtained from transformation of DH5α cells with the ligation product of 600bp PCR products with the EcoRI/BamHI-digested pUC18 dephosphorylated by Antarctic Phosphatase .

Precautions：

Similar to other alkaline phosphatases, Antarctic Phosphatase activity requires the presence of Zn2+ and Mg2+, which can be achieved by adding 1/10 volume of the Antarctic Phosphatase Reaction Buffer (10X) into the reaction.For Antarctic Phosphatase to be active in NEB buffers 1, 2, 3, 4 and CutSmart buffer, Antarctic Phosphatase Reaction Buffer (10X) must be also added.Antarctic Phosphatase activity is enhanced in the presence of monovalent salt ions.Antarctic Phosphatase activity is inhibited by metal chelators such as EDTA, inorganic phosphates and phosphate analogues.The activity of Antarctic Phosphatase decreases in the presence of reducing agents such as DTT and mercaptoethanol.Antarctic Phosphatase should be kept on ice when handling, and should be stored at -20 ℃ immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

1. Dephosphorylation of plasmid DNA in restriction endonuclease reactionsa. Set up the reaction on ice as follows:ComponentQuantityPlasmid DNA1µgEndonuclease Reaction Buffer (10X)2µlNuclease-free waterTo 19µlEndonuclease1μlb. Mix well by gently pipetting or vortex. Centrifuge briefly to collect liquid at the bottom of the centrifuge tube.c. Incubate at 37℃ for 60 minutes or under the recommended conditions following the instructions of the endonuclease.d. Add 1µl of Antarctic Phosphatase (5U/µl) to the reaction and incubate at 37℃ for 15 minutes. The incubation time can be extended to 60 minutes for a thorough dephosphorylation.e. Heat at 75℃ for 5 min to terminate the reaction.f. For subsequent ligation reaction, the products should be purified using the DNA Purification Kit , DNA Gel Recovery Kit , or phenol chloroform extraction followed by ethanol precipitation.2. Dephosphorylation of DNA and RNA a. Set up the reaction on ice as follows:ComponentQuantityDNA or RNA to be dephosphorylated1-5µg (up to 10pmol termini)Antarctic Phosphatase Reaction Buffer (10X)2µlNuclease-free waterTo 19µlAntarctic Phosphatase (5U/μl)1μlb. Mix well by gently pipetting or vortex. Centrifuge briefly to collect liquid at the bottom of the centrifuge tube.c. Incubate at 37℃ for 15 minutes. The incubation time can be extended to 60 minutes for a thorough dephosphorylation.d. Heat at 75℃ for 5 min to terminate the reaction.e. If necessary, the dephosphorylated DNA or RNA can be purified using the DNA Purification Kit , DNA Gel Recovery Kit , or phenol chloroform extraction followed by ethanol precipitation.Note: The above reaction can be used for both 5' protruding and blunt-end DNA.3. Protein dephosphorylationa. Set up the reaction on ice as follows:ComponentQuantityProtein to be dephosphorylated1-10µgAntarctic Phosphatase Reaction Buffer (10X)5µlNuclease-free waterTo 45µlAntarctic Phosphatase (5U/μl)5μlb. Mix well by gently pipetting or vortex. Centrifuge briefly to collect liquid at the bottom of the centrifuge tube.c. Incubate at 37℃ for 60 minutes.d. Add EDTA to a final concentration of 50 mM or sodium vanadate to a final concentration of 10 mM to terminate the reaction.Note: The optimal amount of enzyme and the optimal incubation time at 37℃ should be optimized for different proteins.