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Analysis of DNA‐Protein Interactions Using Proteins Synthesized In Vitro from Cloned Genes

Kevin Struhl¹

¹Harvard Medical School, Boston, Massachusetts

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 12.9

DOI:

10.1002/0471142727.mb1209s24

Online Posting Date:

May, 2001

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Abstract

To detect DNA binding activity, radiolabeled protein is incubated with specific DNA fragments, and protein-DNA complexes are separated from free protein by electrophoresis in native acrylamide gels. Unlike the more conventional mobility shift assay which utilizes ³²P-labeled DNA and unlabeled protein, the assay described here generally utilizes ³⁵S-labeled protein and unlabeled DNA. Major advantages of this method are that any desired mutant protein can be tested for its DNA-binding properties simply by altering the DNA template, and the subunit structure (e.g., dimer, tetramer) can be determined.

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Basic Protocol
Reagents and Solutions
Commentary
Literature Cited

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Materials

Basic Protocol

Materials

Plasmid DNA containing desired binding sites (UNIT 1.6)
³⁵S-labeled protein (UNITS 10.17 & 10.18)
5× binding buffer
10 mg/ml poly(dI-dC)×poly(dI-dC) or other bulk carrier DNA
Loading buffer
45% methanol/10% acetic acid
EN³HANCE (Du Pont NEN)

Additional materials for digesting DNA with restriction endonucleases (UNIT 3.1), phenol extraction and ethanol precipitation (UNIT 2.1A), agarose and nondenaturing polyacrylamide gel electrophoresis (UNITS 2.5A, 2.7, & 12.2), and autoradiography (APPENDIX 3A). For some applications, materials for end-labeling DNA with T4 polyn ucleotide kinase (UNIT 3.9) or Klenow fragment (UNIT 3.5) will be necessary.

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Literature Cited

Literature Cited
	Hope, I.A. and Struhl, K. 1985. GCN4 protein, synthesized in vitro, binds HIS3 regulatory sequences: Implications for general control of amino acid biosynthetic genes in yeast. Cell 43:177-188.
	Hope, I.A. and Struhl, K. 1986. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell 46:885-894.
	Hope, I.A. and Struhl, K. 1987. GCN4, a eukaryotic transcriptional activator protein, binds DNA as a dimer. EMBO J. 6:2781-2784.
	Johnson, A.D. and Herskowitz, I. 1985. A repressor (MAT 2 product) and its operator control a set of cell type specific genes in yeast. Cell 42:237-247.