Promega Corporation

Mapping Intracellular Protein DNA Interactions A more robust efficient alternative...

Mapping Intracellular Protein DNA Interactions A more robust efficient alternative Scientific Poster

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Abstract

Danette D. Hartzell, Marjeta Urh, Natasha Karassina, Georgyi Los, and Keith Wood
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711

Regulation of chromatin structure and gene expression is essential for normal development and cellular growth. Transcriptional events are tightly controlled both spatially and temporally by specific protein:DNA interactions. While significant advances in DNA tiling and microarrays have allowed for genome-wide screening of chromatin recognition sites, current methods to isolate intracellular protein:DNA complexes remain cumbersome and require coimmunoprecipitation, a process inherently subject to capture of nonspecific DNA and proteins. To address these concerns a novel method has been devised for the covalent capture of protein:DNA complexes that does not require the use of antibodies. Proteins of interest are expressed in cells as HaloTag® fusion proteins, crosslinked to DNA, and then captured on HaloLink™ resin, which forms a highly specific, covalent interaction with HaloTag®. Due to the complete covalent linkage established between the resin and the crosslinked protein:DNA complexes, the resin can be stringently washed to remove non-specific DNA and proteins much more effectively than is possible by co-immunoprecipitation. The crosslinks are reversed to release purified DNA fragments from the resin. By improving specificity and reducing background interference during the isolation of protein:DNA complexes, this new methodology effectively increases the signal-to-noise ratio to permit detection of small changes in protein binding within a genome.

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  • Part# PS049
  • Printed in USA.

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