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P4390 Sigma-Aldrich

Polynucleotide Kinase from T4-infected Escherichia coli

10 units/μL, buffered aqueous glycerol solution

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Properties

Related Categories 2.7.x.x Phosphorus containing groups, 2.x.x.x Transferases, Application Index, Biochemicals and Reagents, Enzyme Class Index,
Quality Level   200
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

Description

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.

Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport
WGK Germany 
WGK 3
Flash Point(F) 
Not applicable
Flash Point(C) 
Not applicable

Documents

Certificate of Analysis (COA)

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Certificate of Origin (COO)

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Protocols & Articles

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