Mobility Shift DNA‐Binding Assay Using Gel Electrophoresis

Stephen Buratowski1, Lewis A. Chodosh2

1 Harvard Medical School, Boston, Massachusetts, 2 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 6.8
DOI:  10.1002/0471141755.ph0608s13
Online Posting Date:  August, 2001
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Abstract

DNA‐binding assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence‐specific DNA‐binding proteins. Proteins that bind specifically to an end‐labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein‐DNA complexes. The assay described in this unit can be used to test binding of purified proteins or of uncharacterized factors found in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA‐binding proteins. Three additional protocols describe a competition assay using unlabeled competitor DNA, an antibody supershift assay, and multicomponent gel shift assays.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Mobility Shift Assay
  • Alternate Protocol 1: Competition Mobility Shift Assay
  • Alternate Protocol 2: Antibody Supershift Assay
  • Alternate Protocol 3: Multicomponent Mobility Shift Assays
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Mobility Shift Assay

  Materials
  • 10× electrophoresis buffer, e.g., recipeTAE, recipeTBE, or Tris‐glycine electrophoresis buffer (see recipe)
  • 30% (w/v) ammonium persulfate, prepared fresh
  • N,N,N′,N′‐tetramethylethylenediamine (TEMED)
  • Nondenaturing gel mix (see recipe)
  • Bulk carrier DNA, e.g., poly(dI‐dC)⋅poly(dI‐dC)
  • BSA
  • Protein preparation containing DNA‐binding protein (crude extract or purified fraction)
  • 10× loading buffer with dyes (see recipe)
  • Constant‐temperature water bath
  • Two‐head peristaltic pump
  • 10‐µl glass capillary pipet (optional)
  • Clay‐Adams screw‐top loader (optional)
  • Whatman 3MM filter paper (or equivalent)
  • Additional reagents and equipment for digesting DNA with restriction endonucleases, labeling DNA fragments with Klenow fragment or polynucleotide kinase, agarose and nondenaturing polyacrylamide gel electrophoresis, recovery of DNA from gels, oligonucleotide synthesis, ethanol precipitation, ethidium bromide dot quantitation, and autoradiography (all techniques are described in Ausubel et al., ) and PCR (unit 6.7 in this manual)

Alternate Protocol 1: Competition Mobility Shift Assay

  • Unlabeled specific and nonspecific competitor DNA fragments

Alternate Protocol 2: Antibody Supershift Assay

  • Antibody specific for DNA‐binding protein
  • Nonspecific control antibody
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Figures

Videos

Literature Cited

Literature Cited
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2001. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Carthew, R.W., Chodosh, L.A., and Sharp, P.A. 1985. An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter. Cell 43:439‐448.
   Chodosh, L.A., Carthew, R.W., and Sharp, P.A. 1986. A single polypeptide possesses the binding and activities of the adenovirus major late transcription factor. Mol. Cell. Biol. 6:4723‐4733.
   Fried, M. and Crothers, D.M. 1981. Equilibria and kinetics of lac repressor‐operator interactions by polyacrylamide gel electrophoresis. Nucl. Acids Res. 9:6505‐6525.
   Fried, M. and Crothers, D.M. 1984a. Kinetics and mechanism in the reaction of gene regulatory proteins with DNA. J. Mol. Biol. 172:241‐262.
   Fried, M. and Crothers, D.M. 1984b. Equilibrium studies of the cyclic AMP receptor protein‐DNA interaction. J. Mol. Biol. 172:263‐282.
   Garner, M.M. and Revzin, A. 1981. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: Application to components of the Escherichia coli lactose operon regulatory system. Nucl. Acids Res. 9:3047‐3060.
   Hendrickson, W. and Schleif, R.F. 1984. Regulation of the Escherichia coli L‐arabinose operon studied by gel electrophoresis DNA binding assay. J. Mol. Biol. 174:611‐628.
   Kristie, T.M. and Roizman, B. 1986. a4, the major regulatory protein of herpes simplex virus type 1, is stably and specifically associated with promoter‐regulatory domains of a genes and/or selected viral genes. Proc. Natl. Acad. Sci. U.S.A. 83:3218‐3222.
   Lieberman, P.M. and Berk, A.J. 1994. A mechanism for TAFs in transcriptional activation: Activation domain enhancement of TFIID‐TFIIA‐promoter DNA complex formation. Genes & Dev. 8:995‐1006.
   Riggs, A.D., Suzuki, H., and Bourgeois, S. 1970. Lac repressor‐operator interactions: I. Equilibrium studies. J. Mol. Biol. 48:67‐83.
   Singh, H., Sen, R., Baltimore, D., and Sharp, P.A. 1986. A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature 319:154‐158.
   Staudt, L.M., Singh, H., Sen, R., Wirth, T., Sharp, P.A., and Baltimore, D. 1986. A lymphoid‐specific protein binding to the octamer motif of immunoglobulin genes. Nature 323:640‐643.
   Strauss, F. and Varshavsky, A. 1984. A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome. Cell 37:889‐901.
   Zinkel, S.S. and Crothers, D.M. 1987. DNA bend direction by phase‐sensitive detection. Nature 328:178‐181.
Key References
   Carthew et al., 1985. See above.
  Describes a variation of the mobility shift DNA‐binding assay that is useful in detecting low‐abundance molecules in crude extracts.
   Chodosh et al. 1986. See above.
  Presents a detailed description for measuring kinetic and thermodynamic properties of protein‐DNA interactions using the mobility shift procedure.
   Fried and Crothers 1981. See above.
  Seminal articles on the mobility shift DNA‐binding assay with excellent coverage of many key features of the procedure.
   Garner and Revzin 1981. See above.
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