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

Tsutomu Aoki, Daniel Wolle, Ella Preger Ben Noon, Qi Dai, Eric C. Lai, Paul Schedl

Research output: Contribution to journalArticle

10 Scopus citations

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 chromatinassociated 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.

Original languageEnglish (US)
JournalFly
Volume8
Issue number1
DOIs
StatePublished - 2014

All Science Journal Classification (ASJC) codes

  • Insect Science

Keywords

  • Bi-functional crosslinkers
  • ChIP
  • Chromatin immunoprecipitation
  • DNA binding
  • DSG DSP
  • Elba
  • Formadelhyde
  • Insensitive
  • Insulators

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