title: Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos.
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.
authors.affiliation: Department of Molecular Biology; Princeton University; Princeton, NJ USA.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Aoki
authors.affiliation: Department of Molecular Biology; Princeton University; Princeton, NJ USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wolle
authors.affiliation: Department of Molecular Biology; Princeton University; Princeton, NJ USA.
authors.forename: Ella
authors.initials: E
authors.lastname: Preger-Ben Noon
authors.affiliation: Department of Developmental Biology; Sloan-Kettering Institute; New York, NY USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Dai
authors.affiliation: Department of Developmental Biology; Sloan-Kettering Institute; New York, NY USA.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Lai
authors.affiliation: Department of Molecular Biology; Princeton University; Princeton, NJ USA; Institute of Gene Biology; Russian Academy of Sciences; Moscow, Russian Federation.
authors.forename: Paul
authors.initials: P
authors.lastname: Schedl
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004330:Drosophila; D029721:Drosophila Proteins / Q000302:isolation & purification*; D004625:Embryo, Nonmammalian / Q000187:drug effects*; D013388:Succinimides / Q000494:pharmacology*; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D013388:Succinimides; C096464:disuccinimidyl glutarate; C011240:dithiobis(succinimidylpropionate)
keywords: ChIP; DNA binding; DSG DSP; Elba; Insensitive; bi-functional cross-linkers; chromatin immunoprecipitation; formadelhyde; insulators