T4 Polynucleotide Kinase (PNK)
(ATP: 5'-dephosphopolynucleotide 5'-phosphotransferase E.C.2.7.1.78)
Efficient phosphorylation of nucleic acids for labeling or ligation
Source:
E. coli strain containing a clone of T4 Polynucleotide Kinase(1).
Description:
T4 Polynucleotide Kinase (PNK) catalyzes the transfer of the terminal phosphate of ATP to
5' hydroxyl termini of polynucleotides such as DNA and RNA, oligonucleotides and 3' mononucleotides(2). In the presence of ADP, it will also catalyze the exchange of 5' terminal phosphate groups and ATP. It also possesses a 3' phosphatase(4,5,8,9) and 2',3' cyclic phosphodiesterase activity(3). This enzyme is commonly used to label DNA or RNA with 32P or 33P at 5' ends(6).
Properties:
Molecular Weight: 140 kDa (4 x 33 kDa)
Optimum pH: 7.6 (Tris-HCI)
Optimum Temperature: 37°C
Optimum Mg2+ Concentration: 10mM
Activators: MgCl2, spermidine, dithiothreitol, 2-mercaptoethanol
Inhibitors: Inorganic phosphate, pyrophosphate, ammonium sulfate
Inactivation: 65°C for 10 min
Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating exonucleases, endonucleases and ribonucleases.
Storage Buffer:
50mM Tris-HCl (pH 7.5), 1.0mM DTT, 0.1mM EDTA,50% glycerol.
Assay Conditions:
The reaction mixture (100 μl) contains 50mM Tris-HCI (pH 7.6), 100μM radiolabeled ATP, 10mM MgCl2, 10mM2-mercaptoethanol, 20μM spermidine and DNA substrate. Incubation is at 37°C.
Unit Definition:
One unit is the amount of enzyme required to incorporate 1 nmol of radiolabeled ATP into DNA substrate in 30 min at 37°C.
Concentration:
30 units/μl
Tested User Friendly™ Functional Test:
Phosphorylation of 10 pmol of a 17-mer oligonucleotide with 20 pmol radiolabeled ATP, >50% incorporation of label within 30 min.
Functionally Tested 10X T4 PNK Reaction Buffer (1 ml included): 0.5M Tris-HCl (pH 7.6), 100mM MgCl2, 100mM 2-mercaptoethanol.
T4 PNK Dilution Buffer (1 ml included, PN 71079):
50mM Tris-HCl (pH 8.0).
References:
- MIDGLEY, C. A. AND MURRAY, N. E. (1985) EMBO J. 4, 2695-2703.
- RICHARDSON, C. C. (1981) in The Enzymes, 3rd edition, ed. P. D. Boyer, (Academic Press, New York) 14, 299-314.
- MORSE, D. P. AND BASS, B. L. (1997) Biochemistry 36, 8429-8434.
- CAMERON, V. AND UHLENBECK, O. C. (1977) Biochemistry 16, 5120-5126.
- WANG, L. K. AND SHUMAN, S. (2002) Nucl. Acids Res. 30, 1073-1080.
- MAXAM, A. M. AND GILBERT, W. (1980) Methods in Enzymology 65, 499-560.
- VAN HOUTEN, V., DENKERS, F., VAN DIJK, M., VAN DEN BREKEL, M. AND BRAKENHOFF, R. (1998) Anal. Biochemistry 265, 386-389.
- GALBURT, E., PELLETIER, J., WILSON, G. AND STODDARD, B. (2002) Structure 10, 1249-1260.
- WANG, L. K., LIMA, C. D. AND SHUMAN, S. (2002) EMBO J. 21, 3873-3880.
Applications:
- 5'-labeling of DNA and RNA.
- Mapping termini of RNA transcripts.
- Phosphorylation of oligonucleotides at 5' termini prior to ligation.
- Removal of 3'-phosphoryl groups.
Storage:
Shipped on dry ice. Store at -20°C.
MSDS / Material Safety Data Sheets
T4 Polynucleotide Kinase (PNK)_MSDS
Protocol, Brief
T4 Polynucleotide Kinase (PNK)_Brief Protocol