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Cat. No.: ATPT-1k (for 1KU)

Cat. No.: ATPT-5k (for 5KU)

Description

Antarctic Phosphatase (Thermosensitive) is a heat-sensitive, recombinant phosphatase derived from Antarctic microorganisms. It can catalyze the removal of 5’ or 3’ phosphate groups from DNA, RNA, dNTPs, and rNTPs. After the 5’ end of DNA or RNA is dephosphorylated, it cannot be ligated by ligase, which is commonly used to prevent vector self-ligation, increase the positive rate of gene cloning, and selectively ligate specific sequences.

Antarctic Phosphatase can also remove phosphate groups from serine, threonine, or tyrosine residues in proteins.

Antarctic Phosphatase is a novel alkaline phosphatase that maintains at least 100% activity in various restriction enzyme and PCR buffers from us. Dephosphorylation of various types of DNA 5’ ends can be completed by incubating at 37°C for 15 minutes, and it can be fully inactivated by incubating at 75°C for 5 minutes.

Alkaline Phosphatase (AP/ALP/AKP/ALKP/ALPase/Alk Phos, EC 3.1.3.1) is a class of hydrolases that remove phosphate groups from substrate molecules by hydrolyzing phosphate monoesters, generating phosphate ions and free hydroxyl groups. Its substrates include nucleotides, proteins, and alkaloids, and it is most effective under alkaline conditions. This enzyme is a group of isoenzymes. The commonly used calf intestinal alkaline phosphatase (CIAP/CIP) is widely used for labeling secondary antibodies, ultimately for detecting proteins and nucleic acids, and for dephosphorylating the 5’ and 3’ ends of DNA or RNA, especially for dephosphorylating the 5’ ends of plasmids to prevent self-ligation.

Plasmids digested with restriction endonucleases have 5’ phosphate groups. To prevent plasmid self-ligation during gene cloning, Antarctic Phosphatase can be used to remove the 5’ phosphate groups. Plasmids without 5’ phosphate groups cannot self-ligate.

Main Features

• Rapid dephosphorylation, completed by incubating at 37°C for 15 minutes.
• Rapid inactivation, fully inactivated by incubating at 75°C for 5 minutes.
• Convenient to use, maintaining at least 100% activity in various restriction enzyme and PCR buffers, and can be used simultaneously with plasmid DNA digestion.
• High compatibility, using the same procedure for dephosphorylation of 5’ or 3’ overhangs, blunt ends, and other types of DNA.
• Suitable for subsequent ligation reactions. After dephosphorylation with Antarctic Phosphatase and inactivation by incubating at 75°C for 5 minutes, the DNA can be purified by column purification or gel extraction for subsequent ligation reactions.

Applications

• Prevent vector or DNA fragment self-ligation by removing 5’ phosphate groups.
• Simultaneous restriction enzyme digestion and dephosphorylation of plasmid DNA.
• Prepare DNA or RNA for 5’ end labeling with T4 PNK by dephosphorylating the 5’ ends.
• Remove 5’ phosphate groups from DNA, RNA, rNTPs, and dNTPs.
• Remove dNTPs and pyrophosphate from PCR products.
• Dephosphorylate serine, threonine, and tyrosine residues in proteins.

Source

Recombinant expression in Escherichia coli. Antarctic Phosphatase typically forms homodimers, with each monomer having a molecular weight of 35kD.

Enzyme Activity Definition

One unit of activity is defined as the amount of enzyme required to achieve over 95% inhibition of subsequent self-ligation reactions of 1µg of linearized pUC19 after incubation at 37°C for 30 minutes.

Purity

Free from DNA exonucleases and endonucleases, and RNases.

Inactivation or Inhibition

Fully inactivated by heating at 75°C for 5 minutes. Metal ion chelators like EDTA inhibit Antarctic Phosphatase.

Storage

Store at -20°C.

Precautions

• Antarctic Phosphatase, like other alkaline phosphatases, requires the presence of Zn²⁺ and Mg²⁺ for its enzymatic activity. Adding 1/10 volume of Antarctic Phosphatase Reaction Buffer (10X) to the reaction provides sufficient Zn²⁺ and Mg²⁺ to ensure enzyme activity.
• Antarctic Phosphatase requires the addition of Antarctic Phosphatase Reaction Buffer (10X) to be active in NEB buffers 1, 2, 3, 4, and CutSmart buffer.
• The activity of Antarctic Phosphatase is enhanced in the presence of monovalent salt ions.
• The activity of Antarctic Phosphatase is inhibited by metal ion chelators like EDTA, inorganic phosphate, and phosphate analogs.
• The activity of Antarctic Phosphatase is reduced in the presence of reducing agents such as DTT and β-mercaptoethanol.
• During use, it is recommended to store Antarctic Phosphatase on ice or in an ice bath, and return it to -20°C immediately after use.
• This product is for scientific research use only by professionals and is not intended for clinical diagnosis or treatment, food, or drug use, and should not be stored in ordinary residential areas.
• For your safety and health, please wear a lab coat and disposable gloves when handling.

Phosphatases:

• Alkaline Phosphatase (Thermosensitive)
• Antarctic Phosphatase (Thermosensitive)
• Shrimp Alkaline Phosphatase
• Inorganic Yeast Pyrophosphatase
• Inorganic E.coli Pyrophosphatase
• Thermostable Inorganic Pyrophosphatase

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