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Download 528 MISCELLANEOUS METHODS [32] [32] An Agarose Gel
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528 MISCELLANEOUSMETHODS [32] [32] A n A g a r o s e G e l E l e c t r o p h o r e s i s A s s a y for t h e D e t e c t i o n o f D N A - B i n d i n g A c t i v i t i e s in Y e a s t Cell E x t r a c t s By JUDITH BERMAN, SHLOMO EISENBERG, and BIK-KWOON TYE The gel electrophoresis DNA-binding assay is a simple and versatile method for the quantitative detection and analysis of specific proteinDNA interactions. The history and principles of the assay have been extensively reviewed, l The method is based upon the observation that during gel electrophoresis the mobilities of protein-DNA complexes differ from the mobilities of the uncomplexed components. The method has been used to determine nucleosome composition and structure, 2 and to analyze interactions of purified proteins at the bacterial lactose, 3,4 and Larabinose 5 operons. Recently, the polyacrylamide gel binding assay has also been used to detect specific binding proteins in crude lysates of cells of the African green monkey, 6 Drosophila, 1,7 and Escherichia coli.l The agarose gel electrophoresis DNA-binding assay described here has been used to identify and partially characterize a number of activities, with different DNA-binding specificities, present in yeast cell lysates. While it differs from other recently described a s s a y s 1'6'7 in the use of agarose gels and restricted whole plasmids to screen yeast crude lysates for binding activities, it is based upon the same principles and general approaches as the other assays. The use of agarose gels allows whole plasmids, digested into a number of restriction fragments, to be used as substrates in the assay. Specific DNA-binding activity is observed as a change in the mobility of one specific DNA fragment containing the sequence of interest; nonspecific DNA-binding activities, observed as the altered mobility of all the plasmid fragments, can be minimized by the use of unlabeled carder DNA. 6 Since the method permits relatively large (I kb) fragments to be analyzed, the exact DNA sequence that is bound need not be known, but can be determined by different restriction cuts of the plasmid. Competition studies with plasmid DNA containing the specifically bound DNA fragment can also be used to identify and delimit the binding substrate. I W. Hendrickson, BioTechniques 3, 198 (1985). 2 A. Varshavsky, V. Bakayev, and G. Georgiev, Nucleic Acids Res. 3, 477 (1976). 3 M. Garner and A. Revzin, Nucleic Acids Res. 9, 3047 (1981). 4 M. Fried and D. Crothers, Nucleic Acids Res. 9, 6505 (1981). 5 W. Hendrickson and R. Schleif, J. Mol. Biol. 178, 611 (1984). 6 F. Strauss and A. Varshavsky, Cell 37, 889 (1984). 7 j. Topol, D. M. Ruden, and C. S. Parker, Cell 42, 527 (1985). METHODS IN ENZYMOLOGY, VOL. 155 Copyright © 1987by AcademicPress, Inc. All rights of reproductionin any form reserved.