추천 제품
애플리케이션
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
성분
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.
원리
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
단위 정의
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.
분석 메모
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.
관련 제품
제품 번호
설명
가격
Glutathione S-Transferase from E. coli, recombinant, expressed in E. coli, buffered aqueous solution
Ribonuclease A from bovine pancreas, for molecular biology, ≥70 Kunitz units/mg protein, lyophilized
신호어
Danger
유해 및 위험 성명서
예방조치 성명서
Hazard Classifications
Resp. Sens. 1
Storage Class Code
10 - Combustible liquids
WGK
WGK 3
Flash Point (°F)
Not applicable
Flash Point (°C)
Not applicable
개인 보호 장비
Eyeshields, Gloves, multi-purpose combination respirator cartridge (US)
시험 성적서(COA)
제품의 로트/배치 번호를 입력하여 시험 성적서(COA)을 검색하십시오. 로트 및 배치 번호는 제품 라벨에 있는 ‘로트’ 또는 ‘배치’라는 용어 뒤에서 찾을 수 있습니다.
Polynucleotide kinase exchange reaction: quantitave assay for restriction endonuclease-generated 5'-phosphoroyl termini in DNA.
K L Berkner et al.
The Journal of biological chemistry, 252(10), 3176-3184 (1977-05-25)
Sandeep K Sharma et al.
Nature chemical biology, 6(12), 914-920 (2010-10-19)
Hsp70-Hsp40-NEF and possibly Hsp100 are the only known molecular chaperones that can use the energy of ATP to convert stably pre-aggregated polypeptides into natively refolded proteins. However, the kinetic parameters and ATP costs have remained elusive because refolding reactions have
Problem-solving test: restriction endonuclease mapping.
József Szeberényi
Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology, 39(5), 393-395 (2011-09-29)
Ga Tremblay et al.
Bioanalysis, 3(5), 499-508 (2011-03-11)
Oligonucleotide-based therapeutics are quantified with hybridization assays in biological matrices such as plasma and tissues. Current hybridization methods do not entirely discriminate the parent compound from 5´- or 3´-N-X truncated metabolites. A dual ligation-based hybridization assay was developed to circumvent
Lei Lin et al.
Analytical chemistry, 83(22), 8396-8402 (2011-10-27)
Phosphorylation of DNA with 5'-hydroxyl termini plays a critical role in a majority of normal cellular events, including DNA recombination, DNA replication, and repair of DNA during strand interruption. Determination of nucleotide kinase activity and inhibition is under intense development
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