T4 Polynucleotide Kinase, abbreviated as T4 PNK, is expressed in Escherichia coli. The gene is derived from T4 bacteriophage and can catalyze the phosphorylation of ATP γ- The 5'-hydroxy terminus and 3'-monophosphate nucleosides of the nucleotide chain (double stranded or single stranded DNA or RNA). T4 PNK also possesses 3'-phosphatase activity, which hydrolyze the 3'-phosphate group from the 3'-phosphate terminus of the oligonucleotide, deoxygenated 3'-monophosphate nucleoside, and deoxygenated 3'-diphosphate nucleoside. The T4 PNK can be used for 5' end labeling or phosphorylation of oligonucleotides, DNA, or RNA; the phosphorylation of 5' single nucleotide or removal of the 3' terminal phosphate group. Heating this product at 75°C for 10 minutes or adding EDTA can inactivate it. Ion chelating agents, phosphates, ammonium ions, KCl ≥50 mM, and NaCl can significantly inhibit its activity.
Unit Definition
At 37°C, within 30 minutes, the amount of enzyme required for the transfer of 1 nmol of γ- Phosphate group on ATP to the end of 5′-OH on DNA is defined as 1 active unit.
Quality Control
After multiple column purification, the purity of SDS-PAGE was more than 99%; No contamination of endonuclease, exonuclease, phosphatase and RNase activities was detected.
Precautions
- Because the ammonium salt can strongly inhibit the activity of T4 polynucleotide kinase, the DNA obtained from ammonium salt precipitation cannot be used for the labeling reaction of T4 polynucleotide kinase.
- PEG can promote the rate and efficiency of phosphorylation reaction, and PEG should be added to the exchange reaction system.
- When using, the enzyme should be stored in an ice box or on an ice bath, and should be stored at -20°C immediately after use.
