Beskrivelse
Antarctic Phosphatase (AnP) is a recombinant thermolabile alkaline phosphatase derived from an Antarctic bacterium. It commonly exists as a homodimer and monomer. AnP non-specifically catalyzes the dephosphorylation of 5´- and 3´-terminal phosphate monoesters of DNA and RNA, and hydrolyzes ribonucleoside and deoxyribonucleoside triphosphates (NTPs and dNTPs). The enzyme can be completely and irreversibly inactivated by heating at 75 °C for 5 minutes; therefore, it does not need to be removed prior to ligation or end-labeling. Additionally, this enzyme can also be used for dephosphorylation of serine, threonine, and tyrosine residues on proteins.
Specifications
|
Cat.No. |
14511ES80 / 14511ES90 |
|
Size |
1000 U / 5000 U |
|
Concentration |
5 U/μL |
|
Source |
Recombinant E. coli strain containing the TAB5 AP gene from Antarctic bacterium. |
|
Purity |
≥95% |
|
Tag |
His |
|
Enzyme storage solution |
10 mM Tris-HCl，1 mM MgCl2，0.01 mM ZnCl2，50% Glycerol，pH 7.4 @ 25°C |
|
10×Anp Reaction Buffer |
500 mM Bis-Tris-Propane-HCl , 10 mM MgCl2, 1 mM ZnCl2,(pH 6 @ 25°C) |
Components
|
Components No. |
Name |
14511ES80 |
14511ES90 |
|
14511-A |
Antarctic Phosphatase (5 U/μL) |
200 μL |
1 mL |
|
14511-B |
10×Anp Reaction Buffer |
500 μL |
2×1 mL |
Storage
This product should be stored at -25~-15℃ for 2 years.
Application
- Dephosphorylation of 5´ and 3´ ends of DNA and RNA
- Dephosphorylation of cloning vector DNA to prevent self-ligation
- Dephosphorylation of DNA prior to 5´-end labeling with T4 Polynucleotide Kinase (T4 PNK)
- Removal of residual dNTPs from PCR reactions prior to sequencing or SNP analysis
- Protein dephosphorylation
Instructions
Dephosphorylation of 5' or 3' ends of DNA and RNA
1. Prepare the following reaction mixture and mix thoroughly:
|
Component |
Volume(μL) |
|
DNA or RNA to be dephosphated |
1 µg (1 pmol terminal) |
|
10×Anp Reaction Buffer |
2 μL |
|
Antarctic Phosphatase (5 U/μL) |
1 μL |
|
ddH2O |
To 20 μL |
[Note]: The reaction volume may be scaled up or down proportionally as needed; 1 pmol DNA ≈ 1 μg plasmid (3 kb).
2. Incubate at 37 °C for 15–60 min.
3. Inactivate the reaction by incubating at 75 °C for 5 min.
Dephosphorylation of 5´ ends of plasmid DNA in restriction digestion reactions:
1. Prepare the following reaction mixture and mix thoroughly:
|
Component |
Volume(μL) |
|
Plasmid DNA |
1 µg |
|
Reaction Buffer (10X) for Endonuclease |
2 μL |
|
Endonuclease |
1 μL |
|
ddH2O |
To 20 μL |
2. Perform the restriction digestion under the optimal conditions recommended by the restriction enzyme manufacturer.
3. Add 1 µL of Antarctic Phosphatase (5 U/µL) directly to the digestion reaction and incubate at 37 °C for 15–60 min.
4. Terminate the reaction according to the heat-inactivation protocols for both Antarctic Phosphatase and the restriction enzyme.
Protein Dephosphorylation:
1. Prepare the following reaction mixture and mix thoroughly:
|
Component |
Volume(μL) |
|
Proteins to be dephosphorylated |
1-10 µg |
|
10×Anp Reaction Buffer |
5 μL |
|
Antarctic Phosphatase (5 U/μL) |
5 μL |
|
ddH2O |
To 50 μL |
2. Incubate at 37 °C for 60 min.
3. Stop the dephosphorylation reaction by adding EDTA to a final concentration of 50 mM or sodium orthovanadate to a final concentration of 10 mM.
Notes
1. Activity of thermolabile phosphatase can be enhanced by the addition of appropriate monovalent salts.
2. Metal chelators (e.g., EDTA), inorganic phosphate, and phosphate analogs inhibit thermolabile phosphatase activity. The presence of reducing agents (e.g., DTT, β-mercaptoethanol) reduces enzyme activity.
3. Like most alkaline phosphatases, thermolabile phosphatase is a Zn²⁺- and Mg²⁺-dependent enzyme; the reaction buffer must contain zinc ions.
4. Please operate with lab coats and disposable gloves，for your safety.
5. This product is for research use only.
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