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
Equipment
Procedure
Recipes
Acknowledgments
The use of DNAse I to identify protein bindinging sites on DNA was first described by Galas and Schmitz (1978). Since then it has been exploited and adapted in very many laboratories. Our protocol is based on that in use in the laboratory of Annie Kolb (Colland et al., 2000; Marschall et al., 1998), who introduced the use of potassium glutamate in the binding buffer, which mimics in vivo conditions and increases the affinity of most proteins for their DNA targets. We are extremely grateful to Annie Kolb for her continuous advice and interest. Work in our lab has been funded by the Centre National de Recherche Scientifique (CNRS) to UPR9073 (now renamed FRE3630), by Université Paris 7, Denis Diderot; Agence National de Recherche [(ANR-09-Blanc 0399 (GRONAG)] and by the "Initiative d'Excellence" program from the French state [ANR-11-LBX-0011-01 (DYNAMO)].
References
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