T4 Polynucleotide Kinase (PNK)

Source: E. coli strain containing a clone of T4 Polynucleotide Kinase⁽¹⁾.

Applications:

1. 5'-labeling of DNA and RNA.
2. Mapping termini of RNA transcripts.
3. Phosphorylation of oligonucleotides at 5' termini prior to ligation.
4. Removal of 3'-phosphoryl groups.
   
   

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⁾.

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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^TM 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, PN 70083): 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:

1. MIDGLEY, C. A. AND MURRAY, N. E. (1985) EMBO J. 4, 2695-2703.
2. RICHARDSON, C. C. (1981) in The Enzymes, 3rd edition, ed. P. D. Boyer, (Academic Press, New York) 14, 299-314.
3. MORSE, D. P. AND BASS, B. L. (1997) Biochemistry 36, 8429-8434.
4. CAMERON, V. AND UHLENBECK, O. C. (1977) Biochemistry 16, 5120-5126.
5. WANG, L. K. AND SHUMAN, S. (2002) Nucl. Acids Res. 30, 1073-1080.
6. MAXAM, A. M. AND GILBERT, W. (1980) Methods in Enzymology 65, 499-560.
7. VAN HOUTEN, V., DENKERS, F., VAN DIJK, M., VAN DEN BREKEL, M. AND BRAKENHOFF, R. (1998) Anal. Biochemistry 265, 386-389.
8. GALBURT, E., PELLETIER, J., WILSON, G. AND STODDARD, B. (2002) Structure 10, 1249-1260.
9. WANG, L. K., LIMA, C. D. AND SHUMAN, S. (2002) EMBO J. 21, 3873-3880.
   
   

Storage:
Shipped on dry ice. Store at -20°C.