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Abstract for Gel Shift Assay System
The gel shift, or electrophoretic mobility shift, assay provides a simple and
rapid method for detecting DNA-binding proteins. This method has been used
widely in the study of sequence-specific DNA-binding proteins such as
transcription factors. The assay is based on the observation that complexes of
protein and DNA migrate through a nondenaturing polyacrylamide gel more slowly
than free DNA fragments or double-stranded oligonucleotides. The gel shift assay
is performed by incubating a purified protein, or a complex mixture of proteins
(such as nuclear or cell extract preparations), with a 32P
end-labeled DNA fragment containing the putative protein binding site. The
reaction products are then analyzed on a nondenaturing polyacrylamide gel. The
specificity of the DNA-binding protein for the putative binding site is
established by competition experiments using DNA fragments or oligonucleotides
containing a binding site for the protein of interest or other unrelated DNA
sequences.
Promega has developed Gel Shift Assay Systems that contain target
oligonucleotides, a control extract containing DNA-binding proteins, binding
buffer and reagents for phosphorylating oligonucleotides. The Gel Shift Assay
Core System includes sufficient HeLa nuclear extract to perform 20
control reactions, Gel Shift Binding 5X Buffer, an SP1 Consensus Oligo and an
AP2 Consensus Oligo. This is a reliable system for obtaining experience with gel
shift assays because AP2 binding activity is stable and produces a strong gel
shift.
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