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

互联网2013-12-31

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Abstract
Table of Contents
Materials
Literature Cited

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 ³² P?labeled DNA and unlabeled protein, the assay described here generally utilizes ³⁵ S?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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Reagents and Solutions
Commentary
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Materials

Basic Protocol 1:

Materials

Plasmid DNA containing desired binding sites (unit 1.6 )
³⁵ S‐labeled protein (units 10.17 & 10.18 )
recipe5× binding buffer
10 mg/ml poly(dI‐dC)⋅poly(dI‐dC) or other bulk carrier DNA
recipeLoading buffer
45% methanol/10% acetic acid
EN³ HANCE ( Du Pont NEN)

Additional materials for digesting DNA with restriction endonucleases (unit 3.1 ), phenol extraction and ethanol precipitation (unit 2.1 ), agarose and nondenaturing polyacrylamide gel electrophoresis (units 2.5 , 2.7 , & 12.2 ), and 3.NaNautoradiography ( 3.NaN ). 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 (MATα 2 product) and its operator control a set of cell type specific genes in yeast. Cell 42:237‐247.