Faculty Collaboration Database

Formaldehyde cross-linking for studying nucleosomal dynamics. Methods 1999 Feb;17(2):125-39

Date

03/17/1999

Pubmed ID

10075891

DOI

10.1006/meth.1998.0724

Scopus ID

2-s2.0-0032848160 (requires institutional sign-in at Scopus site)   73 Citations

Abstract

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.

Author List

Jackson V

Author

Vaughn E. Jackson PhD Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

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