Download 528 MISCELLANEOUS METHODS [32] [32] An Agarose Gel

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.