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Fly (Austin). 2014;8(1):43-51. doi: 10.4161/fly.26805. Epub 2013 Dec 13.

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos.

Author information

1
Department of Molecular Biology; Princeton University; Princeton, NJ USA.
2
Department of Developmental Biology; Sloan-Kettering Institute; New York, NY USA.
3
Department of Molecular Biology; Princeton University; Princeton, NJ USA; Institute of Gene Biology; Russian Academy of Sciences; Moscow, Russian Federation.

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 (dithiobis[succinimidyl 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.

KEYWORDS:

ChIP; DNA binding; DSG DSP; Elba; Insensitive; bi-functional cross-linkers; chromatin immunoprecipitation; formadelhyde; insulators

PMID:
24135698
PMCID:
PMC3974894
DOI:
10.4161/fly.26805
[Indexed for MEDLINE]
Free PMC Article

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