title: Chromatin immunoprecipitation assay of brain tissues using Percoll gradient-purified nuclei.
abstract: Protein-DNA interactions are critical to maintain genome stability, DNA replication, chromosome -segregation and to regulate gene expression. Chromatin immunoprecipitation (ChIP) is a powerful technique to study these interactions within living neurons and nervous tissue. In particular, ChIP analysis of chromatin in which protein-DNA interactions are first fixed in situ provides a valuable approach to identify specific transcription factor-DNA interactions and their regulation in the developing nervous system. Here we describe a procedure utilizing Percoll gradient purification of nuclei from fresh brain tissue pre-fixed with formaldehyde for ChIP analysis. This purification protocol provides an enrichment of neuronal nuclei in high yield. We also illustrate the suitability of chromatin prepared from Percoll-purified brain nuclei for ChIP analysis of regulated transcription factor interactions with neuronal gene promoters.
authors.affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Baojin
authors.initials: B
authors.lastname: Ding
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Kilpatrick
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D005557:Formaldehyde / Q000378:metabolism; D051379:Mice; D016133:Polymerase Chain Reaction; D011205:Povidone / Q000494:pharmacology*; D012822:Silicon Dioxide / Q000494:pharmacology*; D016707:Tissue Fixation
chemical_list: D002843:Chromatin; D005557:Formaldehyde; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone