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Chromosome Research

, Volume 24, Issue 2, pp 175–181 | Cite as

ChIP bias as a function of cross-linking time

  • Laura BaranelloEmail author
  • Fedor Kouzine
  • Suzanne Sanford
  • David Levens
Article

Abstract

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins in vivo. Formaldehyde cross-linking of DNA and proteins is a critical step required to trap their interactions inside the cells before immunoprecipitation and analysis. Yet insufficient attention has been given to variables that might give rise to artifacts in this procedure, such as the duration of cross-linking. We analyzed the dependence of the ChIP signal on the duration of formaldehyde cross-linking time for two proteins: DNA topoisomerase 1 (Top1) that is functionally associated with the double helix in vivo, especially with active chromatin, and green fluorescent protein (GFP) that has no known bona fide interactions with DNA. With short time of formaldehyde fixation, only Top1 immunoprecipation efficiently recovered DNA from active promoters, whereas prolonged fixation augmented non-specific recovery of GFP dramatizing the need to optimize ChIP protocols to minimize the time of cross-linking, especially for abundant nuclear proteins. Thus, ChIP is a powerful approach to study the localization of protein on the genome when care is taken to manage potential artifacts.

Keywords

Chromatin immunoprecipitation DNA topoisomerase 1 Green fluorescent protein Formaldehyde cross-linking 

Abbreviations

ChIP

chromatin immunoprecipitation

GFP

green fluorescent protein

NLS

nuclear localization signal

PCR

polymerase chain reaction

qPCR

quantitative PCR

Top1

DNA topoisomerase 1

RNAPII

RNA polymerase II

Notes

Acknowledgments

Our research is supported by the Intramural Research Program of the US National Institutes of Health, Center for Cancer Research of the National Cancer Institute.

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Laura Baranello
    • 1
    Email author
  • Fedor Kouzine
    • 1
  • Suzanne Sanford
    • 1
  • David Levens
    • 1
  1. 1.Laboratory of PathologyNCI/NIHBethesdaUSA

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