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a temporal threshold for formaldehyde crosslinking and fixation甲醛交联时间阈值和固定
A Temporal Threshold for Formaldehyde Crosslinking
and Fixation
. . ´
Lars Schmiedeberg , Pete Skene , Aimee Deaton, Adrian Bird*
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
Abstract
Background: Formaldehyde crosslinking is in widespread use as a biological fixative for microscopy and molecular biology.
An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the
minimum length of time required for an interaction to become fixed has not been determined.
Methodology: Using a unique series of mutations in the DNA binding protein MeCP2, we show that in vivo interactions
lasting less than 5 seconds are invisible in the microscope after formaldehyde fixation, though they are obvious in live cells.
The stark contrast between live cell and fixed cell images illustrates hitherto unsuspected limitations to the fixation process.
We show that chromatin immunoprecipitation, a technique in widespread use that depends on formaldehyde crosslinking,
also fails to capture these transient interactions.
Conclusions/Significance: Our findings for the first time establish a minimum temporal limitation to crosslink chemistry
that has implications for many fields of research.
Citation: Schmiedeberg L, Skene P, Deaton A, Bird A (2009) A Temporal Threshold for Formaldehyde Crosslinking and Fixation. PLoS ONE 4(2): e4636.
doi:10.1371/journal.pone.0004636
Editor: Joshua Z. Rappoport, University of Birmingham, United Kingdom
Received October 29, 2008; Accepted January 27, 2009; Published February 27, 2009
Copyright: 2009 Schmiedeberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, dis
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