A temporal threshold for formaldehyde crosslinking and fixation.
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.


METHODS
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
Our findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research.
Schmiedeberg
Lars
L
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
Skene
Pete
P
Deaton
Aimée
A
Bird
Adrian
A
D000818:Animals; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D005557:Formaldehyde / Q000032:analysis / Q000378:metabolism*; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation
D051783:Methyl-CpG-Binding Protein 2; D005557:Formaldehyde