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T4 Polynucleotide Kinase
$36.00 - $144.00
$160.00
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Cat. No.: T4PK-100 (for 100U)
Cat. No.: T4PK-500 (for 500U)
Description
T4 Polynucleotide Kinase, abbreviated as T4 PNK, is a polynucleotide 5'-hydroxyl kinase that catalyzes the transfer of the γ-phosphate group of ATP to the 5'-hydroxyl of single-stranded or double-stranded DNA, RNA, oligonucleotides, or nucleotides with a 3'-phosphate group. The same reaction can occur with other NTPs: 5'-OH + NTP → 5'-P + NDP.
The aforementioned phosphorylation reaction is reversible. In the absence of ATP and in the presence of ADP, T4 Polynucleotide Kinase can exhibit 5'-phosphatase activity, catalyzing the transfer of the 5'-phosphate group of single-stranded or double-stranded DNA, RNA, oligonucleotides, or nucleotides with a 3'-phosphate group to ADP, forming ATP. The same reaction can occur with other NTPs: 5'-P + NDP → 5'-OH + NTP (optimal pH is around 6.4).
When both ATP and ADP are present in sufficient amounts, T4 Polynucleotide Kinase can catalyze the exchange reaction between the 5'-phosphate group of single-stranded or double-stranded DNA, RNA, oligonucleotides, or nucleotides with a 3'-phosphate group and the γ-phosphate group of ATP. The same reaction can occur with other NTPs: 5'-P + NTP + NDP → 5'-P + NDP + NTP.
T4 Polynucleotide Kinase also exhibits 3'-phosphatase activity, catalyzing the dephosphorylation of 3'-phosphorylated polynucleotides: 3'-P → 3'-OH + Pi (optimal pH is around 5.9).
The kinase activity of T4 Polynucleotide Kinase is near the C-terminus, while the phosphatase activity is near the N-terminus.
Applications
5' end labeling of oligonucleotides, DNA, or RNA for use as probes in Southern, Northern, EMSA, and other assays; gel electrophoresis marker; DNA sequencing primers; PCR primers, etc. It phosphorylates the 5' end of oligonucleotides, DNA, or RNA to ensure the success of subsequent ligation reactions. It catalyzes the 5'-phosphorylation of mononucleotides with a 3'-phosphate group, allowing them to be ligated to the 3' end of DNA or RNA. It can also remove the 3'-phosphate group.
Source
Expressed in Escherichia coli, the expression gene is derived from the T4 bacteriophage.
Activity Definition
The amount of enzyme required to transfer 1 nmol of γ-phosphate from ATP to the 5'-OH end of DNA within 30 minutes at 37°C is defined as one activity unit.
Activity Detection Conditions
100 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM DTT, 0.5 mM 5'-OH DNA, 0.05 mM ATP, 0.1 MBq/ml [γ-33P]-ATP.
Purity
It does not contain DNA endonucleases, exonucleases, or RNAases.
Enzyme Storage Solution
20mM Tris-HCl (pH 7.5), 25mM KCl, 0.1mM EDTA, 2mM DTT, 50% glycerol.
Reaction Buffer A (10X) (for phosphorylation reaction)
500mM Tris-HCl (pH 7.6 at 25℃), 100mM MgCl2, 50mM DTT, 1mM spermidine, 1mM EDTA.
Reaction Buffer B (10X) (for exchange reaction)
500mM imidazole-HCl (pH 6.4 at 25℃), 0.18M MgCl2, 50mM DTT, 1mM spermidine, 1mM EDTA, 1mM ADP.
Inactivation or inhibition
T4 Polynucleotide Kinase can be inactivated by heating at 75°C for 10 minutes or by the addition of EDTA. Metal chelators, phosphates, ammonium ions, KCl, and NaCl concentrations exceeding 50mM can significantly inhibit the activity of T4 Polynucleotide Kinase.
Precautions
- DNA obtained from ammonium salt precipitation should not be used for labeling reactions with T4 Polynucleotide Kinase. Ammonium salts can strongly inhibit the enzyme activity of T4 Polynucleotide Kinase.
- PEG (Polyethylene glycol) can enhance the rate and efficiency of phosphorylation reactions. It should be added to the exchange reaction system.
- The enzyme should be stored in an icebox or on ice during use and immediately placed at -20°C after use for storage.
- This product is intended for scientific research purposes by professionals only. It is not to be used for clinical diagnosis or treatment, food or drug purposes, and should not be stored in residential areas.
- For your safety and health, please wear laboratory attire and disposable gloves when handling.
Storage
Store at -20°C.