Quantitative analysis of macromolecular conformational changes using agarose gel electrophoresis: Application to chromatin folding

Terace M. Fletcher, Philip Serwer, Jeffrey C. Hansen

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Quantitative analysis of chromatin electrophoretic mobility (μ) in agarose gels provides a measure of three structural parameters: average surface electrical charge density, which is proportional to the gel-free μ (μ0), effective radius (Re), and particle deformability [Fletcher, T. M., Krishnan, U., Serwer, P., & Hansen, J. C. (1994) Biochemistry 33, 2226-2233]. To determine whether the intramolecular conformational changes associated with salt-dependent chromatin folding influence these electrophoretic parameters, defined oligonucleosomes were reconstituted from monodisperse tandemly repeated 5S DNA and varying amounts of histone octamers. These oligonucleosomes were subjected to both quantitative agarose gel electrophoresis and analytical velocity ultracentrifugation in buffers containing 0-2 mM MgCl2. Ionic conditions that caused a 40% increase in the oligonucleosome sedimentation coefficient (S20,w) also caused both a 30% decrease in Re and a 60% decrease in the magnitude of the μo. Furthermore, the Mg2+-dependent changes in s20,w, Re, and μo each exhibited the same nonlinear dependence on the degree of nucleosome saturation of the DNA. These data demonstrate that quantitative agarose gel electrophoresis can be used to detect and characterize the process of chromatin folding. In addition, they suggest that this approach can be used for characterization of the conformational dynamics of many other types of macromolecular assemblies, including those systems that are not yet amenable for study by more traditional quantitative biophysical techniques.

Original languageEnglish (US)
Pages (from-to)10859-10863
Number of pages5
JournalBiochemistry
Volume33
Issue number36
StatePublished - 1994

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Agar Gel Electrophoresis
Electrophoresis
Sepharose
Chromatin
Gels
Chemical analysis
Magnesium Chloride
Nucleosomes
Ultracentrifugation
DNA
Biochemistry
Histones
Electrophoretic mobility
Buffers
Formability
Salts
Charge density
Sedimentation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Quantitative analysis of macromolecular conformational changes using agarose gel electrophoresis : Application to chromatin folding. / Fletcher, Terace M.; Serwer, Philip; Hansen, Jeffrey C.

In: Biochemistry, Vol. 33, No. 36, 1994, p. 10859-10863.

Research output: Contribution to journalArticle

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