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The use of nitrocellulose filters to study DNA binding proteins in crude cell lysates: Effect of competing DNA

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Abstract

A nitrocellulose-filter-binding assay system for DNA-protein interactions, suitable for use with crude cell lysates, is described. Such an assay system will detect DNA-binding activities, provided that close attention is paid to the overall concentration of proteins and DNA in the reaction system. The extent of the reduction of generalized DNA-binding by the addition of unlabeled competing DNA is shown to be a function of the source of the competing DNA, since the addition of equal quantities of DNA isolated from different organisms produces drastically different effects. A careful choice of labeled and unlabeled DNA permits preferential binding of sequences from labeled DNA and allows the use of the filter-binding assay as an analytical tool during protein purification.

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Literature Cited

  1. Allen, J. D., Parsons, S. M. 1979. Nitrocellulose filter binding: Quantitation of the histidyl-tRNA-ATP phosphoribosyl-transferase complex. Analytical Biochemistry92:22–30.

    PubMed  Google Scholar 

  2. Apostolakos, D., Birge, E. A. 1979. A thermosensitivepdxJ mutation affecting vitamin B6 biosynthesis inEscherichia coli K-12. Current Microbiology2:39–42.

    Google Scholar 

  3. Birge, E. A., Low, K. B. 1974. Detection of transcribable recombination products following conjugation of Rec+, RecB− and RecC− strains ofEscherichia coli K-12. Journal of Molecular Biology83:447–457.

    PubMed  Google Scholar 

  4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Analytical Biochemistry72:248–254.

    PubMed  Google Scholar 

  5. Bresloff, J. L., Crothers, D. M. 1975. DNA-ethidium reaction kinetics: Demonstration of direct ligand transfer between DNA binding sites. Journal of Molecular Biology95:103–123.

    PubMed  Google Scholar 

  6. Jobe, A., Riggs, A. D., Bourgeois, S. 1972.lac Repressoroperator interaction. V. Characterization of super- and pseudo-wild-type repressors. Journal of Molecular Biology64:181–199.

    PubMed  Google Scholar 

  7. Lin, S.-Y., Riggs, A. D. 1972.lac Repressor binding to nonoperator-DNA: Detailed studies and a comparison of equilibrium and rate competition methods. Journal of Molecular Biology72:671–690.

    PubMed  Google Scholar 

  8. Lin, S.-Y., Riggs, A. D. 1975. The general affinity oflac repressor forE. coli DNA: Implications for gene regulation in prokaryotes and eukaryotes. Cell4:107–111.

    PubMed  Google Scholar 

  9. Pfahl, M. 1978. Effect of DNA denaturants on thelac repressor-operator interaction. Biochimica et Biophysica Acta520:285–290.

    PubMed  Google Scholar 

  10. Riggs, A. D., Suzuki, H., Bourgeois, S. 1970.lac Repressoroperator interaction. Journal of Molecular Biology48:67–83.

    PubMed  Google Scholar 

  11. Taylor, B. L., Utter, N. F. 1974. The removal of nucleic acids from microbial extracts by precipitation with lysozyme. Analytical Biochemistry62:588–591.

    PubMed  Google Scholar 

  12. Waddell, W. J. 1956. A simple ultraviolet spectrophotometric method for the determination of protein. Journal of Laboratory and Clinical Medicine48:311–314.

    PubMed  Google Scholar 

  13. Weideli, H., Gehring, W. J. 1980. A new method for the purification of DNA-binding proteins with sequence specificity. European Journal of Biochemistry104:5–11.

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Botany and Microbiology, Arizona State University, 85287, Tempe, Arizona, USA

    Dennis J. Lye & Edward A. Birge

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  1. Dennis J. Lye
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  2. Edward A. Birge
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Lye, D.J., Birge, E.A. The use of nitrocellulose filters to study DNA binding proteins in crude cell lysates: Effect of competing DNA. Current Microbiology 6, 139–143 (1981). https://doi.org/10.1007/BF01642387

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