Critical parameters for efficient sonication and improved chromatin immunoprecipitation of high molecular weight proteins

Pchelintsev, N. A., Adams, P. D. and Nelson, D. M. (2016) Critical parameters for efficient sonication and improved chromatin immunoprecipitation of high molecular weight proteins. PLoS ONE, 11(1), e0148023. (doi: 10.1371/journal.pone.0148023) (PMID:26821228) (PMCID:PMC4731078)

Abstract

Solubilization of cross-linked cells followed by chromatin shearing is essential for successful chromatin immunoprecipitation (ChIP). However, this task, typically accomplished by ultrasound treatment, may often become a pitfall of the process, due to inconsistent results obtained between different experiments under seemingly identical conditions. To address this issue we systematically studied ultrasound-mediated cell lysis and chromatin shearing, identified critical parameters of the process and formulated a generic strategy for rational optimization of ultrasound treatment. We also demonstrated that whereas ultrasound treatment required to shear chromatin to within a range of 100–400 bp typically degrades large proteins, a combination of brief sonication and benzonase digestion allows for the generation of similarly sized chromatin fragments while preserving the integrity of associated proteins. This approach should drastically improve ChIP efficiency for this class of proteins.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Adams, Professor Peter and Pchelintsev, Dr Nikolay
• Authors: Pchelintsev, N. A., Adams, P. D., and Nelson, D. M.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: PLoS ONE
• Publisher: Public Library of Science
• ISSN: 1932-6203
• ISSN (Online): 1932-6203
• Copyright Holders: Copyright © 2016 Pchelintsev et al.
• First Published: First published in PLoS ONE 11(1):e0148023
• Publisher Policy: Reproduced under a Creative Commons License

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
62558	1	Senescence-associated chromatin changes a barrier to tumor progression.	Peter Adams	Cancer Research UK (CAN-RES-UK)	16566	ICS - EPIGENETICS
51044	4	Epigenetics of Aging and Age-associated diseases : Epigenetic regulation of senescence and aging	Peter Adams	National Institute of Health (USA) (NIH(US))	7P01AG031862-02	RI CANCER SCIENCES