Atomic Force Microscopy of Chromatin

Quénet, Delphine; Dimitriadis, Emilios K.; Dalal, Yamini

doi:10.5772/36722

Cited by 3 publications

(2 citation statements)

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“…Freshly cleaved mica has been traditionally used as it has an atomically flat sheet surface, creating a perfect background for AFM imaging. Untreated, the surface of mica has a negative charge [16, 21] and can effectively bind DNA molecules, provided that salt concentration is low and divalent cations are added to the buffer to bridge the electrostatic interaction with negatively charged phosphate backbone of the DNA. However, under most physiological conditions it does not bind proteins very well.…”

Section: Methodsmentioning

confidence: 99%

“…However, under most physiological conditions it does not bind proteins very well. In order to improve protein deposition, mica can be treated with APTES {(3-Aminopropyl)triethoxysilane} or APS {1-(3-aminopropyl)silatrane} silica compounds that give it a positive surface charge by functionalizing with amino groups [16, 21]. The choice between these two reagents should be determined experimentally, as well as based on feasibility at one’s facility.…”

Section: Methodsmentioning

confidence: 99%

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Tracking Histone Variant Nucleosomes Across the Human Cell Cycle Using Biophysical, Biochemical, and Cytological Analyses

Walkiewicz

Bui

Quénet

et al. 2014

Methods in Molecular Biology

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Histone variants such as H3.3, macroH2A, H2A.Z, and CENP-A are important epigenetic modifiers of the chromatin state in eukaryotic genomes. The centromeric histone H3 variant CENP-A/CENH3 epigenetically marks centromeres and is required for assembly of the kinetochore complex, a region of the chromosome that is responsible for proper genome segregation during mitosis. Several diverse techniques using biochemical, cell biology, and biophysical approaches have been utilized to study the nature of the CENP-A nucleosome across the cell cycle. In this chapter, we describe methods for CENP-A nucleosome purification and separation of CENP-A from other core histones using traditional SDS-PAGE and more resolving techniques such as Triton acid urea (TAU) and two-dimensional gels. We also discuss methods for observation of CENP-A on chromatin fibers using immunofluorescence. Finally, we provide a detailed description of analysis of chromatin structures using atomic force microscopy.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%