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Mobility Shift DNA‐Binding Assay Using Gel Electrophoresis

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The 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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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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Basic Protocol 1: Mobility Shift Assay Materials

10× electrophoresis buffer, e.g., TAE or TBE electrophoresis buffer ( appendix 22 ) or recipeTris‐glycine electrophoresis buffer (see reciperecipe )
30% (w/v) ammonium persulfate, prepared fresh
N,N,N',N' ‐tetramethylethylenediamine (TEMED)
recipeNondenaturing 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 (unit 2.5 )

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 (unit 3.1 ), labeling DNA fragments with Klenow fragment (unit 3.5 ) or polynucleotide kinase (unit 3.10 ), agarose and nondenaturing polyacrylamide gel electrophoresis (unit 2.5 or unit 2.7 ), recovery of DNA from gels (unit 2.6 & unit 12.4 ), oligonucleotide synthesis (unit 2.11 ), PCR (unit 15.1 ), ethanol precipitation (unit 2.1 ), ethidium bromide dot quantitation (unit 2.6 ), and autoradiography ( appendix 3A )

Alternate Protocol 1: Competition Mobility Shift Assay

Unlabeled specific and nonspecific competitor DNA fragments

For this protocol, follow steps 1 to of the protocol 1 to prepare the probe and gel, modify the binding reaction as indicated in step , and proceed with steps through of the protocol 1 . Alternate Protocol 2: Antibody Supershift Assay

Antibody specific for DNA‐binding protein
Nonspecific control antibody

For this protocol, follow steps 1 to of the protocol 1 to prepare the probe and gel, modify the binding reaction as indicated in step , and proceed with steps through of the protocol 1 .

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Figure 12.2.1 Hypothetical autoradiogram of a mobility shift binding experiment analyzing a multicomponent complex (see ). In this experiment, factor A provides sequence‐specific DNA binding. Factors B and C associate with the factor A–DNA complex.

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Figure 12.2.2 Hypothetical autoradiogram of a typical mobility shift DNA‐binding experiment. Radioactive DNA probe and poly(dI‐dC)⋅poly(dI‐dC) were incubated with varying amounts of protein and electrophoresed from top to bottom on a low‐ionic‐strength polyacrylamide gel. The positions of the free probe and bound protein‐DNA complexes are indicated. Lane 1, DNA probe + poly(dI‐dC)⋅poly(dI‐dC); lanes 2 to 5, DNA probe + poly(dI‐dC)⋅poly(dI‐dC) + increasing amounts of protein from crude extract; lane 6, the same as lane 5 except with a large excess of poly(dI‐dC)⋅poly(dI‐dC); lane 7, standard binding reaction; lane 8, standard binding reaction + 50‐fold molar excess of unlabeled probe DNA (specific competitor); lane 9, standard binding reaction + 50‐fold molar excess unlabeled DNA with a sequence unrelated to the probe (nonspecific competitor; also see ).

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Literature Cited

Literature Cited
	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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