Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

PloS One
Nikolay A PchelintsevDavid M Nelson

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.

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Citations

Feb 15, 2017·Journal of Biomedical Materials Research. Part B, Applied Biomaterials·Marek KaweckiAleksandra Kasperczyk
Jan 11, 2019·The Journal of Immunology : Official Journal of the American Association of Immunologists·Rachael L PhilipsVirginia S Shapiro
Sep 13, 2018·Current Protocols in Plant Biology·Jihed ChouarefMaike Stam
Dec 5, 2018·BMC Molecular Biology·Leiah M Luoma, Fred B Berry
Jul 31, 2019·BMC Genomics·Takashi ShimboKatsuto Tamai
Oct 17, 2020·Molecular & Cellular Proteomics : MCP·Guido van Mierlo, Michiel Vermeulen
Feb 9, 2021·Molecular & Cellular Proteomics : MCP·Guido van Mierlo, Michiel Vermeulen
Apr 4, 2020·Molecular Cell·Celeste D RosencranceKyle P Eagen
Aug 20, 2021·Current Protocols·Kasturi MarkandranMichael A Ferenczi
Aug 20, 2021·Nature Communications·Irene Santos-BarriopedroMichiel Vermeulen
May 22, 2020·Langmuir : the ACS Journal of Surfaces and Colloids·Jenna A DombroskiMichael R King

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Methods Mentioned

BETA
immunoprecipitation
PCR
ChIP
pull-down
electrophoresis
ChIP-seq

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