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Analysis of Protein-DNA Equilibria by Native Gel Electrophoresis

  • Chapter
Protein Interactions

Part of the book series: Protein Reviews ((PRON,volume 5))

Abstract

The electrophoretic mobility-shift assay (EMSA) is widely used to detect protein–nucleic acid interactions [for representative reviews, see Garner and Revzin (1986), Chodosh (1988), Fried (1989), Carey (1991), Lane et al. (1992), Fried and Garner (1998)]. A particular strength of this method is its ability to detect the simultaneous binding of several proteins to a single molecule of nucleic acid (Figure 14.1), or one (or more) protein(s) to several nucleic acids. Such interactions are of interest because nucleic acid complexes containing large number of proteins play central roles in important cellular transactions including (but not limited to) DNA replication, recombination and repair, and RNA transcription and processing. Although mobility-shift assays are often used for qualitative purposes, under appropriate conditions they can provide quantitative data for the determination of binding stoichiometries, affinities, and kinetics.

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References

  • Amersham Biosciences (2002). Typhoon User’s Guide Vol. 3.0, sections 6-1-6-22.

    Google Scholar 

  • Bading, H. (1988). Determination of the molecular weight of DNA-bound protein(s) responsible for gel electrophoretic mobility shift of linear DNA fragments examplified with purified viral myb protein. Nucl Acids Res 16:5241-5248.

    PubMed  CAS  Google Scholar 

  • Bain, D. L. and Ackers, G. K. (1998). A quantitative cryogenic gel-shift technique for analysis of protein-DNA binding. Anal Biochem 258:240-245.

    PubMed  CAS  Google Scholar 

  • Barkley, M. (1981). Salt dependence of the kinetics of the lac repressor-operator interaction: role of nonoperator deoxyribonucleic acid in the association reaction. Biochemistry 20:3833-3842.

    PubMed  CAS  Google Scholar 

  • Berger, R., Duncan, M. R., and Berman, B. (1993). Nonradioactive gel mobility shift assay using chemiluminescent detection. Biotechniques 15:650-652.

    PubMed  CAS  Google Scholar 

  • Berman, J., Eisenberg, S., and Tye, B.-K. (1987). An agarose gel electrophoresis assay for the detection of DNA binding activities in yeast cell extracts. Methods Enzymol 155:528-537.

    PubMed  CAS  Google Scholar 

  • Bloomfield, V. A. and Lim, T. K. (1978). Quasi-elastic light scattering. Methods Enzymol 48:415-494.

    PubMed  CAS  Google Scholar 

  • olton, A. E. and Hunter, W. M. (1973). The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J 133:529-239.

    Google Scholar 

  • Burchard, W. (1992). Static and Dynamic Light Scattering. In Harding, S. E., Sattelle, D. B. and Bloomfield, V. A. (eds), Laser Light Scattering in Biochemistry. The Royal Society of Chemistry, Cambridge, pp. 3-22.

    Google Scholar 

  • Burnette, W. N. (1981). ‘‘Western blotting’’: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112: 195-203.

    PubMed  CAS  Google Scholar 

  • Calero, M. and Ghiso, J. (2005). Radiolabeling of amyloid-beta peptides. Methods Mol Biol 299: 325-348.

    PubMed  CAS  Google Scholar 

  • Carey, J. (1988). Gel retardation at low pH resolves trp repressor-DNA complexes for quantitative study. Proc Natl Acad Sci U S A 85:975-979.

    PubMed  CAS  Google Scholar 

  • Carey, J. (1991). Gel retardation. Methods Enzymol 208:103-117.

    PubMed  CAS  Google Scholar 

  • Chahla, M., Wooll, J., Laue, T. M., Nguyen, N., and Senear, D. F. (2003). Role of protein-protein bridging interactions on cooperative assembly of DNA-bound CRP-CytR-CRP complex and regulation of the Escherichia coli CytR regulon. Biochemistry 42:3812-3825.

    PubMed  CAS  Google Scholar 

  • Chen, H. and Chang, G. D. (2001). Simultaneous immunoblotting analysis with activity gel electrophor-esis in a single polyacrylamide gel. Electrophoresis 22:1894-1899.

    PubMed  CAS  Google Scholar 

  • Chodosh, L. A. (1988). Mobility shift DNA-binding assay using gel electrophoresis. In Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D, Seidman, J. G., Smith, J. A., and Struhl, K. (eds), Current Protocols in Molecular Biology. John Wiley and Sons, New York, pp. 12.12.11-12.12.10.

    Google Scholar 

  • Clark, W. A., Izotova, L., Philipova, D., Wu, W., Lin, L., and Pestka, S. (2002). Site-specific 32P-labeling of cytokines, monoclonal antibodies, and other protein substrates for quantitative assays and therapeutic application. Biotechniques 33 Suppl:S76-S87.

    Google Scholar 

  • Crothers, D. M. and Drak, J. (1992). Global features of DNA structure by comparative gel electrophor-esis. Methods Enzymol 212:46-71.

    PubMed  CAS  Google Scholar 

  • Crothers, D. M., Gartenberg, M. R., and Schrader, T. E. (1991). DNA bending in protein-DNA complexes. Methods Enzymol 208:118-146.

    PubMed  CAS  Google Scholar 

  • Daugherty, M. A. and Fried, M. G. (2003). Analysis of transcription factor interactions at sedimentation equilibrium. In: Adhya S. and Garges, S. (eds), Methods in Enzymol, v. 370: RNA Polymerases and Associated Factors, Part C. Elsevier, New York, pp. 349-369.

    Google Scholar 

  • de Boer, A. R., Letzel, T., Lingeman, H., and Irth, H. (2005). Systematic development of an enzymatic phosphorylation assay compatible with mass spectrometric detection. Anal Bioanal Chem 381:647-655.

    PubMed  CAS  Google Scholar 

  • Delucchi, A. B., Jensen, K. A., and Chan, W. K. (2003). Synthesis of 32P-labelled protein probes using a modified thioredoxin fusion protein expression system in Escherichia coli. Biomol Eng 20:1-5.

    PubMed  CAS  Google Scholar 

  • Dhavan, G. M., Crothers, D. M., Chance, M. R., and Brenowitz, M. (2002). Concerted binding and bending of DNA by Escherichia coli integration host factor. J Mol Biol 315:1027-1037.

    PubMed  CAS  Google Scholar 

  • Ferber, M. J. and Maher, L. Jr. (1997). Quantitating oligonucleotide affinities for duplex DNA: footprint-ing vs electrophoretic mobility shift assays. Anal Biochem 244:312-320.

    PubMed  CAS  Google Scholar 

  • Fickert, T. and Muller-Hill, B. (1992). How lac repressor finds lac operator in vitro. J Mol Biol 226:59-68.

    PubMed  CAS  Google Scholar 

  • Fried, M. G. (1989). Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis 10:366-376.

    PubMed  CAS  Google Scholar 

  • Fried, M. G. and Bromberg, J. L. (1997). Factors that affect the stability of protein-DNA complexes during gel electrophoresis. Electrophoresis 18:6-11.

    PubMed  CAS  Google Scholar 

  • Fried, M. G. and Crothers, D. M. (1981). Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucl Acids Res 9:6505-6525.

    PubMed  CAS  Google Scholar 

  • Fried, M. G. and Crothers, D. M. (1984a). Equilibrium studies of the cyclic AMP receptor protein-DNA interaction. J Mol Biol 172:241-262.

    PubMed  CAS  Google Scholar 

  • Fried, M. G. and Crothers, D. M. (1984b). Kinetics and mechanism in the reactions of gene regulatory proteins with DNA. J Mol Biol 172:263-282.

    PubMed  CAS  Google Scholar 

  • Fried, M. G. and Daugherty, M. A. (1998). Electrophoretic analysis of multiple protein-DNA inter-actions. Electrophoresis 19:1247-1253.

    PubMed  CAS  Google Scholar 

  • Fried, M. G. and Garner, M. M. (1998). The electrophoretic mobility shift assay (emsa) for detection and analysis of protein-DNA interactions. In: Tietz, D. (ed.) Molecular Biology Methods and Applications. Elsevier, New York, pp. 239-271.

    Google Scholar 

  • Fried, M. G., Kanugula, S., Bromberg, J. L., and Pegg, A. E. (1996). DNA binding mechanisms of O6 -alkylguanine-DNA alkyltransferase: stoichiometry and effects of DNA base composition and secondary structures on complex stability. Biochemistry 35:15295-15301.

    PubMed  CAS  Google Scholar 

  • Fried, M. G. and Liu, G. (1994). Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis. Nucl Acids Res 22:5054-5059.

    PubMed  CAS  Google Scholar 

  • Fried, M. G., Stickle, D., Vossen, K., Adams, C., MacDonald, D., and Lu, P. (2002). Role of macromol-ecular hydration in lac repressor-DNA interactions. J Biol Chem 277:50676-50682.

    PubMed  CAS  Google Scholar 

  • Gaidamakov, S. A., Gorshkova, I. I., Schuck, P., Steinbach, P. J., Yamada, H., Crouch, R. J., and Cerritelli, S. M. (2005). Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain. Nucl Acids Res 33:2166-2175.

    PubMed  CAS  Google Scholar 

  • 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 system. Nucl Acids Res 9:3047-3060.

    PubMed  CAS  Google Scholar 

  • Garner, M. M. and Revzin, A. (1982). Stoichiometry of catabolite activator protein=adenosine cyclic3’,5’-monophosphate interactions at the lac promoter of Escherichia coli. Biochemistry 21: 6032-6036.

    PubMed  CAS  Google Scholar 

  • Garner, M. M. and Revzin, A. (1986). The use of gel electrophoresis to detect and study nucleic acid-protein interactions. Trends Biol Sci 11:395-396.

    CAS  Google Scholar 

  • Gavigan, S. A., Nguyen, T., Nguyen, N., and Senear, D. F. (1999). Role of multiple CytR binding sites on cooperativity, competition, and induction at the Escherichia coli udp promoter. J Biol Chem 274:16010-16019.

    PubMed  CAS  Google Scholar 

  • Gerstle, J. T. and Fried, M. G. (1993). Measurement of binding kinetics using the gel electrophoresis mobility shift assay. Electrophoresis 14:725-731.

    PubMed  CAS  Google Scholar 

  • Gotoh, O., Wada, A., and Yabuki, S. (1979). Salt-concentration dependence of melting profiles of lambda phage DNAs: evidence for long-range interactions and pronounced end effects. Biopolymers 18:805-824.

    PubMed  CAS  Google Scholar 

  • Hall, D. and Minton, A. P. (2003). Macromolecular crowding: qualitative and semiquantitative successes, quantitative challenges. Biochim Biophys Acta 1649:127-139.

    PubMed  CAS  Google Scholar 

  • 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 178:611-628.

    PubMed  CAS  Google Scholar 

  • Heyduk, T. and Lee, J. C. (1990). Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction. Proc Natl Acad Sci U S A 87:1744-1748.

    PubMed  CAS  Google Scholar 

  • Hill, T. L. (1985). Cooperativity Theory in Biochemistry. Springer-Verlag, New York.

    Google Scholar 

  • Howard, V. J., Belyaeva, T. A., Busby, S. J. W., and Hyde, E. I. (2002). DNA binding of the transcription activator protein MelR from Escherichia coli and its C-terminal domain. Nucl Acids Res 30:2692-2700.

    PubMed  CAS  Google Scholar 

  • Hsieh, M. and Brenowitz, M. (1997). Comparison of the DNA association kinetics of the Lac repressor tetramer, its dimeric mutant LacIadi, and the native dimeric Gal repressor. J Biol Chem 272: 22092-22096.

    PubMed  CAS  Google Scholar 

  • Hsiung, S. K., Lin, C. H., and Lee, G. B. (2005). A microfabricated capillary electrophoresis chip with multiple buried optical fibers and microfocusing lens for multiwavelength detection. Electrophor-esis 26:1122-1129.

    CAS  Google Scholar 

  • Huang, C. Y. (1982). Determination of binding stoichiometry by the continuous variation method: the Job plot. Methods Enzymol 87:509-525.

    PubMed  CAS  Google Scholar 

  • Hudson, J. M. and Fried, M. G. (1990). Co-operative interactions between the catabolite gene activator protein and the lac repressor at the lactose promoter. J Mol Biol 214:381-396.

    PubMed  CAS  Google Scholar 

  • Jing, D., Agnew, J., Patton, W. F., Hendrickson, J., and Beechem, J. M. (2003). A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels. Proteomics 3:1172-1180.

    PubMed  CAS  Google Scholar 

  • Jing, D., Beechem, J. M., and Patton, W. F. (2004). The utility of a two-color fluorescence electrophoretic mobility shift assay procedure for the analysis of DNA replication complexes. Electrophoresis 25:2439-2446.

    PubMed  CAS  Google Scholar 

  • Kash, J. C. and Menon, K. M. (1998). Identification of a hormonally regulated luteinizing hormone= human chorionic gonadotropin receptor mRNA binding protein. Increased mRNA binding during receptor down-regulation. J Biol Chem 273:10658-10664.

    PubMed  CAS  Google Scholar 

  • Kozlov, A. G. and Lohman, T. M. (2002). Kinetic mechanism of direct transfer of Escherichia coli SSB tetramers between single-stranded DNA molecules. Biochemistry 41:11611-11627.

    PubMed  CAS  Google Scholar 

  • Krämer, H., Amouyal, M., Nordheim, A., and Muller-Hill, B. (1988). DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor. EMBO J 7:547-556.

    PubMed  Google Scholar 

  • Krämer, H., Niemoller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B., and Muller-Hill, B. (1987). Lac repressor forms loops with linear DNA carrying two suitably spaced lac operators. EMBO J 6:1481-1491.

    PubMed  Google Scholar 

  • Lane, D., Prentki, P., and Chandler, M. (1992). Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev 56:509-528.

    PubMed  CAS  Google Scholar 

  • L’Annunziata, M. F. (2005). Application Note CIA 002: Cerenkov Counting of 32 P—Instrument Performance Data. Packard Instrument Co., Meriden, CT, p. 5.

    Google Scholar 

  • Laue, T. M. (1995). Sedimentation equilibrium as a thermodynamic tool. Methods Enzymol 259: 427-452.

    PubMed  CAS  Google Scholar 

  • Lobell, R. B. and Schlief, R. F. (1990). DNA looping and unlooping by AraC protein. Science 250: 528-532.

    PubMed  CAS  Google Scholar 

  • Lobell, R. and Schleif, R. (1991). AraC-DNA looping: orientation and distance-dependent loop breaking by the cyclic AMP receptor protein. J Mol Biol 218:45-54.

    PubMed  CAS  Google Scholar 

  • Lomholt, B. and Frederiksen, S. (1987). Detection of a few picograms of DNA on polyacrylamide gels by silver staining. Anal Biochem 164:146-149.

    PubMed  CAS  Google Scholar 

  • Long, K. S. and Crothers, D. M. (1995). Interaction of human immunodeficiency virus type 1 tat-derived peptides with TAR RNA. Biochemistry 34:8885-8895.

    PubMed  CAS  Google Scholar 

  • Lorand, L., Parameswaran, K. N., Velasco, P. T., Hsu, L. K., and Siefring, G. E. Jr. (1983). New colored and fluorescent amine substrates for activated fibrin stabilizing factor (Factor XIIIa) and for transglutaminase. Anal Biochem 131:419-425.

    PubMed  CAS  Google Scholar 

  • Mackun, K. and Downard, K. M. (2003). Strategy for identifying protein-protein interactions of gel-separated proteins and complexes by mass spectrometry. Anal Biochem 318:60-70.

    PubMed  CAS  Google Scholar 

  • Maillet, I., Lagniel, G., Perrot, M., Boucherie, H., and Labarre, J. (1996). Rapid identification of yeast proteins on two-dimensional gels. J Biol Chem 271:10263-10270.

    PubMed  CAS  Google Scholar 

  • Maxam, A. and Gilbert, W. S. (1977). A new method for sequencing DNA. Proc Natl Acad Sci U S A 74:560-565.

    PubMed  CAS  Google Scholar 

  • McGhee, J. and von Hippel, P. H. (1974). Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice. J Mol Biol 86:469-489.

    PubMed  CAS  Google Scholar 

  • Means, G. E. (1977). Reductive alkylation of amino groups. Methods Enzymol 47:469-478.

    PubMed  CAS  Google Scholar 

  • Means, G. E. and Feeney, R. E. (1995). Reductive alkylation of proteins. Anal Biochem 224:1-16.

    PubMed  CAS  Google Scholar 

  • Melançon, P., Burgess, R. R., and Record, M. T. Jr. (1983). Direct evidence for the preferential binding of Escherichia coli RNA polymerase holoenzyme to the ends of deoxyribonucleic acid restriction fragments. Biochemistry 22:5169-5176.

    PubMed  Google Scholar 

  • Melcher, K. and Xu, H. E. (2001). Gal80-Gal80 interaction on adjacent Gal4p binding sites is required for complete GAL gene repression. EMBO J 20:841-851.

    PubMed  CAS  Google Scholar 

  • Milev, S., Bosshard, H. R., and Jelesarov, I. (2005). Enthalpic and entropic effects of salt and polyol osmolytes on site-specific protein-DNA association: The Integrase Tn916-DNA complex. Biochemistry 44:285-293.

    PubMed  CAS  Google Scholar 

  • Mills, M., Arimondo, P. B., Lacroix, L., Garestier, T., Klump, H., and Mergny, J. L. (2002). Chemical modification of the third strand: differential effects on purine and pyrimidine triple helix formation. Biochemistry 41:357-366.

    PubMed  CAS  Google Scholar 

  • Mucha, P., Szyk, A., Rekowski, P., and Barciszewski, J. (2002). Structural requirements for conserved Arg52 residue for interaction of the human immunodeficiency virus type 1 trans-activation respon-sive element with trans-activator of transcription protein (49-57). Capillary electrophoresis mobility shift assay. J Chromatogr A 968:211-220.

    PubMed  CAS  Google Scholar 

  • Mukhopadhyay, J., Mekler, V., Kortkhonjia, E., Kapanidis, A. N., Ebright, Y. W., and Ebright, R. H. (2003). Fluorescence resonance energy transfer (FRET) in analysis of transcription-complex struc-ture and function. Methods Enzymol 371:144-159.

    PubMed  CAS  Google Scholar 

  • Muller, J., Oehler, S. and Muller-Hill, B. (1996). Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator. J Mol Biol 257:21-29.

    PubMed  CAS  Google Scholar 

  • Nagata, H., Tabuchi, M., Hirano, K., and Baba, Y. (2005). Microchip electrophoretic protein separation using electroosmotic flow induced by dynamic sodium dodecyl sulfate-coating of uncoated plastic chips. Electrophoresis 26:2687-2691.

    PubMed  CAS  Google Scholar 

  • Naritsin, D. B. and Lyubchenko, Y. L. (1991). Melting of oligodeoxynucleotides with various structures. J Biomol Struct Dyn 8:813-825.

    PubMed  CAS  Google Scholar 

  • Nordheim, A. and Meese, K. (1988). Topoisomer gel retardation: detection of anti-Z-DNA antibodies bound to Z-DNA within supercoiled DNA minicircles. Nucleic Acids Res 16:21-37.

    PubMed  CAS  Google Scholar 

  • Oehler, S., Eismann, E. R., Krämer, H., and Muller-Hill, B. (1990). The three operators of the lac operon cooperate in repression. EMBO J 9:973-979.

    PubMed  CAS  Google Scholar 

  • Olmsted, M. C., Anderson, C. F., and Record, M. T. (1989). Monte Carlo description of oligoelectrolyte properties of DNA oligomers: range of the end effect and the approach of molecular and thermo-dynamic properties to the polyelectrolyte limits. Proc Natl Acad Sci U S A 86:7766-7770.

    PubMed  CAS  Google Scholar 

  • Park, S. H. and Raines, R. T. (1997). Green fluorescent protein as a signal for protein-protein inter-actions. Protein Sci 6:2344-2349.

    PubMed  CAS  Google Scholar 

  • Park, S. H. and Raines, R. T. (2004). Fluorescence gel retardation assay to detect protein-protein interactions. Methods Mol Biol 261:155-160.

    PubMed  CAS  Google Scholar 

  • Peeni, B. A., Conkey, D. B., Barber, J. P., Kelly, R. T., Lee, M. L., Woolley, A. T., and Hawkins, A. R. (2005). Planar thin film device for capillary electrophoresis. Lab Chip 5:501-505.

    PubMed  CAS  Google Scholar 

  • Perri, S. D., Helinski, D. R., and Toukdarian, A. (1991). Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2. J Biol Chem 266:12536-12543.

    PubMed  CAS  Google Scholar 

  • Plenert, M. L. and Shear, J. B. (2003). Microsecond electrophoresis. Proc Natl Acad Sci U S A 100: 3853-3857.

    PubMed  CAS  Google Scholar 

  • Qu, L., Li, X., Wu, G., and Yang, N. (2005). Efficient and sensitive method of DNA silver staining in polyacrylamide gels. Electrophoresis 26:99-101.

    PubMed  CAS  Google Scholar 

  • Rasimas, J. J., Pegg, A. E., and Fried, M. G. (2003). DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase. Effects of protein and DNA alkylation on complex stability. J Biol Chem 278:7973-7980.

    PubMed  CAS  Google Scholar 

  • Record, M. T., Anderson, C. F., and Lohman, T. M. (1978). Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity. Q Rev Biophys 11:103-178.

    PubMed  CAS  Google Scholar 

  • Record, M. T., Anderson, C. F., Mossing, M., and Roe, J.-H. (1985). Ions as regulators of protein-nucleic acid interactions in vitro and in vivo. Adv Biophys 20:109-135.

    PubMed  CAS  Google Scholar 

  • Roder, K. and Schweizer, M. (2001). Running-buffer composition influences DNA-protein and protein-protein complexes detected by electrophoretic mobility-shift assay (EMSA). Biotechnol Appl Biochem 33:209-214.

    PubMed  CAS  Google Scholar 

  • Rodgers, J. T., Patel, P., Hennes, J. L., Bolognia, S. L., and Mascotti, D. P. (2000). Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays. Anal Biochem 277:254-259.

    PubMed  CAS  Google Scholar 

  • Rouault, T. A., Hentze, M. W., Haile, D. J., Harford, J. B., and Klausner, R. D. (1989). The iron-responsive element binding protein: a method for the affinity purification of a regulatory RNA-binding protein. Proc Natl Acad Sci U S A. 86:5768-5772.

    PubMed  CAS  Google Scholar 

  • Rye, H. S., Drees, B. L., Nelson, H. C., and Glazer, A. N. (1993). Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat shock transcription factor. J Biol Chem 268:25229-25238.

    PubMed  CAS  Google Scholar 

  • Scatchard, G. (1949). The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51: 660-672.

    CAS  Google Scholar 

  • Schleif, R. (1992). DNA looping. Annu Rev Biochem 61:199-223.

    PubMed  CAS  Google Scholar 

  • Senear, D. F. and Brenowitz, M. (1991). Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel mobility-shift assay. J Biol Chem 266:13661-13671.

    PubMed  CAS  Google Scholar 

  • Senear, D. F., Dalma-Weiszhausz, D. D., and Brenowitz, M. (1993). Effects of anomalous migration and DNA to protein ratios on resolution of equilibrium constants from gel mobility-shift assays. Electrophoresis 14:704-712.

    PubMed  CAS  Google Scholar 

  • Seyfried, N. T., Blundell, C. D., Day, A. J., and Almond, A. (2005). Preparation and application of biologically active fluorescent hyaluronan oligosaccharides. Glycobiology 15:303-312.

    PubMed  CAS  Google Scholar 

  • Shimba, N., Yamada, N., Yokoyama, K., and Suzuki, E. (2002). Enzymatic labeling of arbitrary proteins. Anal Biochem 301:123-127.

    PubMed  CAS  Google Scholar 

  • Sidorova, N. Y. and Rau, D. C. (2004). Differences between EcoRI nonspecific and ‘‘star’’ sequence complexes revealed by osmotic stress. Biophys J 87:2564-2576.

    PubMed  CAS  Google Scholar 

  • Smith, C. L. and Peterson, C. L. (2003). Coupling tandem affinity purification and quantitative tyrosine iodination to determine subunit stoichiometry of protein complexes. Methods 31:104-109.

    PubMed  CAS  Google Scholar 

  • Stern, R. V. and Frieden, E. (1993). Partial purification of the rat erythrocyte ceruloplasmin receptor monitored by an electrophoresis mobility shift assay. Anal Biochem 212:221-228.

    PubMed  CAS  Google Scholar 

  • Straney, D. C. and Crothers, D. M. (1985). Intermediates in transcription initiation from the E. coli lac UV5 promoter. Cell 43:449-459.

    PubMed  CAS  Google Scholar 

  • Torizawa, T., Shimizu, M., Taoka, M., Miyano, H., and Kainosho, M. (2004). Efficient production of isotopically labeled proteins by cell-free synthesis: a practical protocol. J Biomol NMR 30:311-312.

    PubMed  CAS  Google Scholar 

  • Towbin, H., Staehelin, T., and Gordon, J. (1989). Immunoblotting in the clinical laboratory. J Clin Chem Clin Biochem 27:495-501.

    PubMed  CAS  Google Scholar 

  • Towbin, H., Staehelin, T., and Gordon, J. (1992). Electrophoretic transfer of proteins from polyacryla-mide gels to nitrocellulose sheets: procedure and some applications. Biotechnology 24:145-149.

    PubMed  CAS  Google Scholar 

  • Tsodikov, O. V., Holbrook, J. A., Shkel, I. A., and Record, M. T. Jr. (2001). Analytic binding isotherms describing competitive interactions of a protein ligand with specific and nonspecific sites on the same DNA oligomer. Biophys J 81:1960-1969.

    PubMed  CAS  Google Scholar 

  • Ueda, C. T. and Roberts, R. W. (2004). Analysis of a long-range interaction between conserved domains of human telomerase RNA. RNA 10:139-147.

    PubMed  CAS  Google Scholar 

  • Vallone, P. M. and Benight, A. S. (2000). Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in four 40 base pair deoxyoligonucleotides. Biochemistry 39:7835-7846.

    PubMed  CAS  Google Scholar 

  • Vossen, K. M. and Fried, M. G. (1997). Sequestration stabilizes Lac repressor-DNA complexes during gel electrophoresis. 245:85-92.

    CAS  Google Scholar 

  • Vossen, K. M., Stickle, D. F., and Fried, M. G. (1996). The mechanism of CAP-lac repressor binding cooperativity at the E. coli lactose promoter. J Mol Biol 255:44-54.

    PubMed  CAS  Google Scholar 

  • Wang, S., Huber, P. W., Cui, M., Czarnik, A. W., and Mei, H.-Y. (1998). Binding of Neomycin to the TAR Element of HIV-1 RNA Induces Dissociation of Tat Protein by an Allosteric Mechanism. Biochemistry 37:5549-5557.

    PubMed  CAS  Google Scholar 

  • Wu, H. M. and Crothers, D. M. (1984). Identification of the locus of sequence-directed and protein-induced DNA bending. Nature 308:509-513.

    PubMed  CAS  Google Scholar 

  • Wu, Z. L., Zhang, L., Beeler, D. L., Kuberan, B., and Rosenberg, R. D. (2002). A new strategy for defining critical functional groups on heparan sulfate. FASEB J 16:539-545.

    PubMed  CAS  Google Scholar 

  • Wyman, J. and Gill, S. J. (1990). Binding and Linkage. University Science Books, Mill Valley, CA.

    Google Scholar 

  • Xian, J. and Harrington, M. G. (1998). Mobility shift electrophoresis of protein-DNA complexes by capillary electrophoresis. In Tietz, D. (ed.), Nucleic Acid Electrophoresis. Springer, Berlin, pp. 272-291.

    Google Scholar 

  • Xuan, X. and Li, D. (2005). Band-broadening in capillary zone electrophoresis with axial temperature gradients. Electrophroesis 26:166-175.

    CAS  Google Scholar 

  • Zerby, D. and Lieberman, P. M. (1997). Functional analysis of TFIID-activator interaction by magne-sium-agarose gel electrophoresis. Methods 12:217-223.

    PubMed  CAS  Google Scholar 

  • Zimmerman, S. B. and Minton, A. P. (1993). Macromolecular crowding: biochemical, biophysical and physiological consequences. Annu Rev Biophys Biomol Struct 22:27-65.

    PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA

    Claire A. Adams

  2. Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA

    Michael G. Fried

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  1. Claire A. Adams
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  2. Michael G. Fried
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Editors and Affiliations

  1. National Institutes of Health, Bethesda, MD, 20892, USA

    Peter Schuck

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Adams, C.A., Fried, M.G. (2007). Analysis of Protein-DNA Equilibria by Native Gel Electrophoresis. In: Schuck, P. (eds) Protein Interactions. Protein Reviews, vol 5. Springer, Boston, MA. https://doi.org/10.1007/978-0-387-35966-3_14

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