US EPA

1512385 

Journal Article 

Formaldehyde cross-linking for studying nucleosomal dynamics 

Jackson, V 

1999 

1 

Methods
ISSN: 1046-2023
EISSN: 1095-9130 

17 

2 

125-139 

English 

10075891 

10.1006/meth.1998.0724 

WOS:000079291100005 

http://://WOS:000079291100005
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Methods are described for the utilization of formaldehyde as a reversible cross-linking agent for the characterization of protein-protein and protein-DNA interactions. The methods include a description of procedures to: (1) isolate and characterize transcriptionally active chromatin from cells cross-linked with formaldehyde; (2) study histone mobility during replication and transcription by the characterization of the formaldehyde-cross-linked histone octamer that is isolated from cells labeled with density-labeled amino acids; and (3) cross-link the in vitro reconstituted histone-DNA complex in order to maintain its structural state during subsequent characterization. Included in these methods are procedures for a second dimensional analysis of protein-protein cross-links in which the monomer components are electrophoretically resolved in the second dimension. The methods also include procedures to selectively reverse protein-DNA cross-links while maintaining the protein-protein cross-links. Potential artifacts are also discussed; i.e., data are presented which indicate that the helical pitch of DNA can be altered if the ionic strength is not properly controlled. The stability of the cross-linked nucleosome in the presence of altered pH or salt/urea concentrations is described in order to indicate that there are limitations to procedures that can be used for the subsequent characterization of the cross-linked complex. 

Cross-Linking Reagents/ metabolism; Electrophoresis, Polyacrylamide Gel; Formaldehyde/ metabolism; Histones/metabolism; Hydrogen-Ion Concentration; Magnesium/pharmacology; Molecular Biology/ methods; Nucleosomes/ metabolism; Ribonuclease, Pancreatic/metabolism; Sonication; Time Factors