The product is supplied in a solution of 50% (v/v) glycerol, 20 mM Tris-HCl, pH 7.5, with 25 mM KCl, 2 mM DTT, 0.1 mM EDTA, and 0.1 uM ATP.
P4390
Polynucleotide Kinase from T4-infected Escherichia coli
10 units/μL, buffered aqueous glycerol solution
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A propos de cet article
Numéro CAS:
UNSPSC Code:
12352204
NACRES:
NA.53
MDL number:
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Polynucleotide Kinase from T4-infected Escherichia coli, 10 units/μL, buffered aqueous glycerol solution
grade
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
Quality Level
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Cet article | SRE0005 | D8276 | P4850 |
|---|---|---|---|
| grade for molecular biology | grade for molecular biology | grade for molecular biology | grade for molecular biology |
| concentration 10 units/μL | concentration ≥10 mg/mL protein, ≥800 unit/mL | concentration ~3,000 units/mL | concentration ≥10 mg/mL, ≥800 units/mL |
| form buffered aqueous glycerol solution | form buffered aqueous glycerol solution | form buffered aqueous glycerol solution | form buffered aqueous glycerol solution |
| mol wt 33 kDa | mol wt 28.93 kDa | mol wt 103 kDa | mol wt 28.93 kDa |
| shipped in wet ice | shipped in wet ice | shipped in wet ice | shipped in - |
| storage temp. −20°C | storage temp. 2-8°C | storage temp. −20°C | storage temp. 2-8°C |
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
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.
Biochem/physiol Actions
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
Other Notes
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.
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.
signalword
Danger
hcodes
pcodes
Hazard Classifications
Resp. Sens. 1
Classe de stockage
10 - Combustible liquids
wgk
WGK 3
flash_point_f
Not applicable
flash_point_c
Not applicable
ppe
Eyeshields, Gloves, multi-purpose combination respirator cartridge (US)
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V Cameron et al.
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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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