The detection of DNA-binding proteins by protein blotting (original) (raw)
Abstract
A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed.
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- Adolph K. W., Cheng S. M., Laemmli U. K. Role of nonhistone proteins in metaphase chromosome structure. Cell. 1977 Nov;12(3):805–816. doi: 10.1016/0092-8674(77)90279-3. [DOI] [PubMed] [Google Scholar]
- Alberts B. M., Amodio F. J., Jenkins M., Gutmann E. D., Ferris F. L. Studies with DNA-cellulose chromatography. I. DNA-binding proteins from Escherichia coli. Cold Spring Harb Symp Quant Biol. 1968;33:289–305. doi: 10.1101/sqb.1968.033.01.033. [DOI] [PubMed] [Google Scholar]
- Berezney R., Coffey D. S. The nuclear protein matrix: isolation, structure, and functions. Adv Enzyme Regul. 1976;14:63–100. doi: 10.1016/0065-2571(76)90008-x. [DOI] [PubMed] [Google Scholar]
- Cattan D., Bourgoin D. Interactions between DNA and hen's egg white lysozyme. Effects of composition and structural changes of DNA, ionic strength and temperature. Biochim Biophys Acta. 1968 Jun 18;161(1):56–67. doi: 10.1016/0005-2787(68)90294-3. [DOI] [PubMed] [Google Scholar]
- Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
- Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
- Eisenman R. N., Vogt V. M. The biosynthesis of oncovirus proteins. Biochim Biophys Acta. 1978 Apr 6;473(3-4):187–239. doi: 10.1016/0304-419x(78)90014-8. [DOI] [PubMed] [Google Scholar]
- Fox T. O., Bates S. E., Vito C. C., Wieland S. J. Carrier protein effects on DNA-cellulose chromatography of putative steroid receptors. J Biol Chem. 1979 Jun 25;254(12):4963–4966. [PubMed] [Google Scholar]
- Goodwin G. H., Johns E. W. Isolation and characterisation of two calf-thymus chromatin non-histone proteins with high contents of acidic and basic amino acids. Eur J Biochem. 1973 Dec 3;40(1):215–219. doi: 10.1111/j.1432-1033.1973.tb03188.x. [DOI] [PubMed] [Google Scholar]
- Hinkle D. C., Chamberlin M. J. Studies of the binding of Escherichia coli RNA polymerase to DNA. I. The role of sigma subunit in site selection. J Mol Biol. 1972 Sep 28;70(2):157–185. doi: 10.1016/0022-2836(72)90531-1. [DOI] [PubMed] [Google Scholar]
- Jones O. W., Berg P. Studies on the binding of RNA polymerase to polynucleotides. J Mol Biol. 1966 Dec 28;22(2):199–209. doi: 10.1016/0022-2836(66)90126-4. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Levy W B., Wong N. C., Dixon G. H. Selective association of the trout-specific H6 protein with chromatin regions susceptible to DNase I and DNase II: possible location of HMG-T in the spacer region between core nucleosomes. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2810–2814. doi: 10.1073/pnas.74.7.2810. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Renart J., Reiser J., Stark G. R. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3116–3120. doi: 10.1073/pnas.76.7.3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
- Riggs A. D., Bourgeois S. On the assay, isolation and characterization of the lac repressor. J Mol Biol. 1968 Jul 14;34(2):361–364. doi: 10.1016/0022-2836(68)90260-x. [DOI] [PubMed] [Google Scholar]
- Riggs A. D., Suzuki H., Bourgeois S. Lac repressor-operator interaction. I. Equilibrium studies. J Mol Biol. 1970 Feb 28;48(1):67–83. doi: 10.1016/0022-2836(70)90219-6. [DOI] [PubMed] [Google Scholar]
- Rosenthal A. L., Lacks S. A. Nuclease detection in SDS-polyacrylamide gel electrophoresis. Anal Biochem. 1977 May 15;80(1):76–90. doi: 10.1016/0003-2697(77)90627-3. [DOI] [PubMed] [Google Scholar]
- Sato S., Hutchinson C. A., 3rd, Harris J. I. A thermostable sequence-specific endonuclease from Thermus aquaticus. Proc Natl Acad Sci U S A. 1977 Feb;74(2):542–546. doi: 10.1073/pnas.74.2.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weber K., Kuter D. J. Reversible denaturation of enzymes by sodium dodecyl sulfate. J Biol Chem. 1971 Jul 25;246(14):4504–4509. [PubMed] [Google Scholar]
- Weintraub H., Van Lente F. Dissection of chromosome structure with trypsin and nucleases. Proc Natl Acad Sci U S A. 1974 Oct;71(10):4249–4253. doi: 10.1073/pnas.71.10.4249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weisbrod S., Weintraub H. Isolation of a subclass of nuclear proteins responsible for conferring a DNase I-sensitive structure on globin chromatin. Proc Natl Acad Sci U S A. 1979 Feb;76(2):630–634. doi: 10.1073/pnas.76.2.630. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wray W., Stubblefield E. A new method for the rapid isolation of chromosomes, mitotic apparatus, or nuclei from mammalian fibroblasts at near neutral pH. Exp Cell Res. 1970 Mar;59(3):469–478. doi: 10.1016/0014-4827(70)90656-7. [DOI] [PubMed] [Google Scholar]
- Yarus M., Berg P. On the properties and utility of a membrane filter assay in the study of isoleucyl-tRNA synthetase. Anal Biochem. 1970 Jun;35(2):450–465. doi: 10.1016/0003-2697(70)90207-1. [DOI] [PubMed] [Google Scholar]