P4390

Sigma-Aldrich

Polynucleotide Kinase from T4-infected Escherichia coli

10 units/μL, buffered aqueous glycerol solution

CAS Number:
Enzyme Commission number:
MDL number:
NACRES:
NA.53

Quality Level

grade

for molecular biology

form

buffered aqueous glycerol solution

mol wt

33 kDa

concentration

10 units/μL

foreign activity

Endonuclease and exonuclease, none detected

shipped in

wet ice

storage temp.

−20°C

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Application

Suitable for:
  • Sequencing or nucleic acid tagging (DNA and RNA) by 5′-end labeling
  • 5′ phosphorylation of oligonucleotides
  • Removal of 3′-phosphate groups from phosphorylpolynucleotides

Components

T4 Polynucleotide Kinase is supplied in a solution of 50% glycerol (v/v), 20 mM Tris-HCl (pH 7.5), 25 mM KCl, 2mM DTT, 0.1 mM EDTA, and 0.1 μM ATP.

Principle

Polynucleotide kinase catalyses a "forward reaction" transfer of the γ-phosphate of ATP to the 5′ hydroxyl terminus of single- and double-stranded nucleic acids (DNA and RNA) and 3′-nucleoside monophosphates. In exchange reactions containing ADP, the enzyme will catalyze the exchange of 5′-terminal phosphate groups and ATP. The 3′-phosphatase activity enables the enzyme to remove 3′-phosphoryl groups from phosphorylpolynucleotides.
1. Forward reaction: Transfer of the labeled γ-phosphate from [γ-32P]-ATP to the free 5′-hydroxyl group of the substrate.
5′-HO-DNA + [γ-32P]-ATP → 5′-32PO-DNA + ADP.
Substrates that do not have a free 5′-hydroxyl require prior dephosphorylation by alkaline phosphatase.
2. Exchange reaction: First, the terminal 5′-phosphate is transferred from the substrate to ADP present in the reaction mixture. Then, the labeled γ-phosphate from [γ-32P]-ATP is transferred to the free hydroxyl group of the substrate.
5′-PO-DNA + ADP → 5′-HO-DNA + ATP
5′-HO-DNA + [γ-32P]-ATP → 5′-32PO-DNA + ADP

Unit Definition

One unit catalyzes the transfer of one nanomole of 32P to the 5′-end of micrococcal nuclease-treated DNA in 30 min. at 37 °C. Transfer is detected as incorporation into acid-insoluble material.

Analysis Note

Activity is determined in a reaction mixture containing 40 mM Tris-HCl (pH 7.5), with 10 mM MgCl2, 5 mM dithiothreitol, 0.5 mM 5′-OH polynucleotide ends, and mM [γ-32P]-ATP.

Pictograms

Health hazard

Signal Word

Danger

Hazard Statements

Precautionary Statements

hazcat

Resp. Sens. 1

storage_class_code

10 - Combustible liquids

WGK Germany

WGK 3

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Personal Protective Equipment

dust mask type N95 (US),Eyeshields,Gloves

Certificate of Analysis

Certificate of Origin

V Cameron et al.
Biochemistry, 16(23), 5120-5126 (1977-11-15)
The purification of T4 polynucleotide kinase results in the copurification of an activity which will specifically remove the 3'-terminal phosphate from a variety of deoxyribonucleotides and ribonucleotides in the absence of ATP. This phosphatase activity requires magnesium, has a pH...
Wenhe Wu et al.
Chemical communications (Cambridge, England), 47(4), 1201-1203 (2010-11-26)
We designed a single-fluorophore-tagged hairpin-structured nano-beacon probe by using a superquencher, graphene oxide (GO), based on which a new method for the analysis of DNA phosphorylation detection was developed.
Saranya Siribal et al.
Molecular and biochemical parasitology, 180(1), 1-7 (2011-08-09)
Polynucleotide kinase/phosphatase (PNKP) is a bifunctional enzyme that can phosphorylate the 5'-OH termini and dephosphorylate the 3'-phosphate termini of DNA. It is a DNA repair enzyme involved in the processing of strand break termini, which permits subsequent repair proteins to...
Audun Hanssen-Bauer et al.
Environmental and molecular mutagenesis, 52(8), 623-635 (2011-07-26)
XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as...
Nigel J Jones
Methods in molecular biology (Clifton, N.J.), 817, 183-206 (2011-12-08)
32P-postlabelling is a technique originally described by Kurt Randerath and colleagues for the sensitive detection of damage produced in DNA by reactive chemicals or genotoxins. The procedure essentially entails the enzymatic digestion of DNA to nucleoside 3'-monophosphates which are then...

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