脱氧核糖核酸酶I 足迹法识别蛋白结合位点

Isabelle Gaugué; Dominique Bréchemier-Baey; Jacqueline Plumbridge

doi:10.21769/BioProtoc.824

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

DNase I Footprinting to Identify Protein Binding Sites

脱氧核糖核酸酶I 足迹法识别蛋白结合位点

IG Isabelle Gaugué

DB Dominique Bréchemier-Baey

JP Jacqueline Plumbridge email

发布: 2013年07月20日第3卷第14期 DOI: 10.21769/BioProtoc.824 浏览次数: 22811

评审: Fanglian He

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Molecular Microbiology, featuring study using the protocol.

Sep 2012

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

³²P-磷酸盐与DNA寡核苷酸3′端的连接

Joshua C. Cofsky and Jennifer A. Doudna

2020年10月20日 4159 阅读

用原子力显微镜描述DNA解旋酶PriA与停止的DNA复制叉的相互作用

Yaqing Wang [...] Yuri L. Lyubchenko

2021年03月05日 4053 阅读

一种基于凝胶的检测dsDNA蛋白易位的方法

Christiane Brugger and Alexandra M. Deaconescu

2021年07月20日 3324 阅读

Abstract

DNase I footprinting is used to precisely localise the position that a DNA binding protein, e.g. a transcription factor, binds to a DNA fragment. A DNA fragment of a few hundred bp is labelled at one end and then incubated with the proteins suspected to bind. After a limited digestion with DNase I, the reaction is quenched, DNA is precipitated and analysed on a denaturing polyacrylamide gel. This protocol uses ³²P-radioactively labeled DNA.

Materials and Reagents

Oligonucleotides (usually 20-30 mer) to amplify a suitable fragment (100-400 bp) encompassing the region to be tested for protein binding ability
Plasmid DNA carrying the cloned required region to use as template for the PCR amplification
[γ-³²P] ATP (3,000 Ci/mmole, 30 μCi = 3 μl/labeling ) (e.g. NEN, catalog number: BLU502A)
Polynucleotide kinase (PNK) (e.g. Biolabs, catalog number: M0201 )
Agarose
Purified protein (or enriched crude bacterial extracts see below)
DNase I (e.g. Sigma-Aldrich, catalog number: D5025 )
Phenol
Chloroform
Herring sperm DNA (e.g. Sigma-Aldrich, catalog number: D6898 ; Roche, catalog number: 223 646 )
BSA (e.g. Biolabs, catalog number: B9001 )
Acrylamide
Urea
DTTP (e.g. Biolabs, catalog number: N0447 )
Taq polymerase (5 units/μl) (e.g. Biolabs, catalog number: M0267 )
DNA Marker (e.g. 100 bp ladder, Biolabs, catalog number: N3231 )
Antarctic alkaline phosphatase (Biolabs, catalog number: M0296 )
MspI (Biolabs, catalog number: N3032 )
40% Acrylamide stock (19:1 acrylamide: bis acrylamide) (e.g. Euromedex, catalog number: EU0076-C)
TBE buffer
Binding buffer (see Recipes)
DNase I dilution buffer (see Recipes)
DNase I stop buffer (see Recipes)
DNase I stock (see Recipes)
Loading formamide dyes (see Recipes)
Denaturing Sequencing gel (6% acrylamide) (see Recipes)
Hepes-Glutamate (see Recipes)

Equipment

Suitable space for working with ³²P radioactivity
Image quantification apparatus (e.g. Typhoon GE Healthcare Life Sciences; X-ray film and developing materials)
PCR machine
Small horizontal agarose gel apparatus
Transilluminator (preferably 365 nM)
Apparatus for running a 30 cm sequencing gel (e.g. Model S2 Vertical sequencing apparatus, now sold by Biometra)
Power supply capable of producing 2,000 volts and 60 watts)
Geiger counter to monitor for radioactivity and any contamination.
Heating block at 90 °C
Gel drying apparatus

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Gaugué, I., Bréchemier-Baey, D. and Plumbridge, J. (2013). DNase I Footprinting to Identify Protein Binding Sites. Bio-protocol 3(14): e824. DOI: 10.21769/BioProtoc.824.