Gel Shift Assay Systems

The gel shift or electrophoretic mobility shift assay provides a simple and rapid method for detecting DNA-binding proteins. This method is widely used to study 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 ...

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The gel shift or electrophoretic mobility shift assay provides a simple and rapid method for detecting DNA-binding proteins. This method is widely used to study 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 unlabeled DNA fragments or oligonucleotides containing a binding site for the protein of interest or other unrelated DNA sequences.

The Core System (Cat.# E3050) includes HeLa Nuclear Extract and SP1 and AP2 Consensus Oligos that can be used as positive controls and serve as a reliable system for obtaining experience with gel shift assays. In addition, the Core System contains T4 Polynucleotide Kinase and Kinase 10X Buffer for labeling oligonucleotides as well as Gel Shift Binding 5X Buffer. Cat.# E3300 contains all of the above plus consensus oligos for AP1, OCT1, CREB, NF-κB, and TFIID.

Features - Benefits

• Positive Controls: The Gel Shift Assay Core System includes a HeLa Nuclear Extract and consensus oligonucleotides for AP2 and SP1.
• Versatile: Oligonucleotides can be 5´ end-labeled and used as protein-specific probes or used as unlabeled oligonucleotides in competition assays.

Applications

• Assay for DNA-binding proteins that bind to transcription factor consensus sequences or DNA sequences of your choice.

For more information, see the Protocols & Applications Guide.

References

1. Ausubel, F. et al. (1989) In: Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates and Wiley-Interscience, Unit 12.2.

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