[9] Quantitative DNase footprint titration: A method for studying protein-DNA interactions

Brenowitz, Michael; Senear, Donald F.; Shea, Madeline A.; Ackers, Gary K.

doi:10.1016/0076-6879(86)30011-9

Cited by 389 publications

(328 citation statements)

References 28 publications

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“…A third limitation is that the electrophoretic mobility of a complex provides little direct information about the location of the nucleic acid sequences that are occupied by protein. This information is available from nuclease and chemical footprinting assays that can be performed independently of EMSA or in concert with it [35][36][37][38] . Finally, the time resolution of the current assay is defined by the interval required for manual solution handling.…”

Section: Advantages and Limitations Of Emsamentioning

confidence: 99%

“…The most widely used alternative assays are nitrocellulose filter-binding 40,41 and footprinting 35,36,42 .…”

Section: Alternatives To Emsamentioning

confidence: 99%

“…Footprinting assays 35,51 exploit the observation 42 that a protein bound to a specific nucleic acid sequence will interfere with the chemical or enzymatic modification of that sequence. A large number of chemical and enzymatic agents are available for this purpose.…”

Section: Alternatives To Emsamentioning

confidence: 99%

“…This is an important advantage over non-equilibrium assays such as EMSA. Variants of the footprinting assay optimized for quantitative detection of binding have been described 35,52 and time-resolved methods have been developed that allow the analysis of binding kinetics as well as equilibria 53,54 .…”

Section: Alternatives To Emsamentioning

confidence: 99%

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Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007

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The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32 P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

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Section: Advantages and Limitations Of Emsamentioning

confidence: 99%

“…The most widely used alternative assays are nitrocellulose filter-binding 40,41 and footprinting 35,36,42 .…”

Section: Alternatives To Emsamentioning

confidence: 99%

Section: Alternatives To Emsamentioning

confidence: 99%

Section: Alternatives To Emsamentioning

confidence: 99%

See 2 more Smart Citations

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007

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“…DNase I footprinting analysis was performed as described by Brenowitz et al (1986). Primers shown in Fig.…”

Section: Dnase I Footprint Analysismentioning

confidence: 99%

MIDA1 is a sequence specific DNA binding protein with novel DNA binding properties

2000

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Background: Id proteins not only regulate cell differentiation negatively, but they also promote growth and apoptosis. To know the mechanism of how Id regulates cell fate, we previously isolated an Idassociating protein, MIDA1, which positively regulates cell growth. Its predicted amino acid sequence contains tryptophan-mediated repeats (Tryp-med repeats) similar to the DNA binding region of the cMyb oncoprotein. We determined whether MIDA1 can bind to DNA in a sequence speci®c manner by PCR-assisted binding site selection.

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Kooperative Haarnadel-Dimere für die Erkennung von DNA durch Pyrrol/Imidazol-Polyamide

1998

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[9] Quantitative DNase footprint titration: A method for studying protein-DNA interactions

Cited by 389 publications

References 28 publications

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

MIDA1 is a sequence specific DNA binding protein with novel DNA binding properties

Kooperative Haarnadel-Dimere für die Erkennung von DNA durch Pyrrol/Imidazol-Polyamide

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