P4390
Polynukleotid-Kinase aus T4-infizierten E. coli
10 units/μL, buffered aqueous glycerol solution
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About This Item
CAS-Nummer:
MDL-Nummer:
UNSPSC-Code:
12352204
NACRES:
NA.53
Empfohlene Produkte
Qualität
for molecular biology
Qualitätsniveau
Form
buffered aqueous glycerol solution
Mol-Gew.
33 kDa
Konzentration
10 units/μL
Fremdaktivität
Endonuclease and exonuclease, none detected
Versandbedingung
wet ice
Lagertemp.
−20°C
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Anwendung
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
Komponenten
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.
Prinzip
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
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
Einheitendefinition
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.
Hinweis zur Analyse
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.
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Signalwort
Danger
H-Sätze
P-Sätze
Gefahreneinstufungen
Resp. Sens. 1
Lagerklassenschlüssel
10 - Combustible liquids
WGK
WGK 3
Flammpunkt (°F)
Not applicable
Flammpunkt (°C)
Not applicable
Persönliche Schutzausrüstung
Eyeshields, Gloves, multi-purpose combination respirator cartridge (US)
Analysenzertifikate (COA)
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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
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
A role in true-late gene expression for the T4 bacteriophage 5' polynucleotide kinase 3' phosphatase.
K Sirotkin et al.
Journal of molecular biology, 123(2), 221-233 (1978-08-05)
Eduardo Paredes et al.
Methods (San Diego, Calif.), 54(2), 251-259 (2011-03-01)
Advances in RNA nanotechnology will depend on the ability to manipulate, probe the structure and engineer the function of RNA with high precision. This article reviews current abilities to incorporate site-specific labels or to conjugate other useful molecules to RNA
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
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