Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009;4(2):e4636.
doi: 10.1371/journal.pone.0004636. Epub 2009 Feb 27.

A temporal threshold for formaldehyde crosslinking and fixation

Lars Schmiedeberg  1 , Pete Skene, Aimée Deaton, Adrian Bird
Affiliations

A temporal threshold for formaldehyde crosslinking and fixation

Lars Schmiedeberg et al. PLoS One. 2009.

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Major differences in nuclear localisation of MeCP2-GFP in living versus paraformaldehyde-fixed mouse fibroblasts.
All MeCP2 mutants (labelled left) localized to nuclear foci corresponding to peri-centromeric heterochromatin in living cells, but many showed diffuse nuclear staining in the same cells after paraformaldehyde-fixation.
Figure 2
Figure 2. Inverse relationship between MeCP2 residence time on heterochromatin and the ability to crosslink this interaction by formaldehyde.
A) In vivo residence times on heterochromatin of MeCP2 mutants were determined by FRAP. Mutants with a residence time above 5 seconds were 100% localized in fixed cells, whereas those with shorter residence times localized partially or not at all. B) Immunoprecipitation of formaldehyde-crosslinked MeCP2 is inefficient when the residence time is below 4 seconds. The mutants in order from left to right in panels A and B are: R168X, R106W, R111G, D97E, T158M, L100V, R133C and wildtype. Error bars on both axes correspond to ±standard deviation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Solomon MJ, Larsen PL, Varshavsky A. Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell. 1988;53:937–47. - PubMed
    1. Solomon MJ, Varshavsky A. Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. Proc Natl Acad Sci U S A. 1985;82:6470–4. - PMC - PubMed
    1. Gilmour DS, Rougvie AE, Lis JT. Protein-DNA cross-linking as a means to Determine the distribution of proteins on DNA in vivo. Methods in Cell Biology. 1991;35:369–381. - PubMed
    1. Orlando V, Strutt H, Paro R. Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods. 1997;11:205–14. - PubMed
    1. Lewis JD, Meehan RR, Henzel WJ, Maurer-Fogy I, Jeppesen P, et al. Purification, sequence and cellular localisation of a novel chromosomal protein that binds to methylated DNA. Cell. 1992;69:905–914. - PubMed

Publication types