Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos |
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Authors: | Tsutomu Aoki Daniel Wolle Ella Preger-Ben Noon Qi Dai Eric C Lai Paul Schedl |
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Institution: | 1.Department of Molecular Biology; Princeton University; Princeton, NJ USA;2.Department of Developmental Biology; Sloan-Kettering Institute; New York, NY USA;3.Institute of Gene Biology; Russian Academy of Sciences; Moscow, Russian Federation |
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Abstract: | Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobissuccinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking. |
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Keywords: | chromatin immunoprecipitation ChIP formadelhyde bi-functional cross-linkers insulators DSG DSP DNA binding Elba Insensitive |
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