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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PDF or HTML at Wiley Online LibraryTable of Contents
- 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
Alternate Protocol 1: Competition Mobility Shift Assay
Alternate Protocol 2: Antibody Supershift Assay
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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. View Image -
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 ). View Image
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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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