[Abstract]  [Full Text]  [PDF]

Robin Ricke¹ and Anja-Katrin Bielinsky^1*

¹ Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota. Minneapolis, MN 55455. USA.

* To whom correspondence should be addressed: Anja-Katrin Bielinsky, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota. 321 Church Street SE, 6-155 Jackson Hall, Minneapolis, MN 55455. USA. Phone: 612-624-2469. Fax: 612-625-2163. Email: bielinsk@cbs.umn.edu

Biol. Proced. Online 2005;7:60-69. doi:10.1251/bpo106
Submitted: March 22, 2005; Accepted: April 14, 2005; Published: May 09, 2005.

Indexing terms: Chromatin; Cross-linking Reagents; Formaldehyde; Histones; Immunoprecipitation.

Abbreviations: ChIP, Chromatin Immunoprecipitation; HAA, Histone Association Assay; Mcm10, Minichromosome Maintenance Protein 10; Orc2, Origin Recognition Complex protein 2; WCE, Whole Cell Extract.

The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and non-chromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes.