Experimental setup for the study of immobilized single nucleosomes using total internal reflection fluorescence

被引：0

作者：

Kudryashova K.S. ^{[1
,2
]}

Chertkov O.V. ^{[1
,2
]}

Ivanov Y.O. ^{[1
]}

Studitskiy V.M. ^{[1
,3
]}

Feofanov A.V. ^{[1
,2
]}

机构：

[1] Department of Biology, Moscow State University, Moscow

[2] Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow

[3] Laboratory of Cancer Epigenetics, Fox Chase Cancer Research Center, 333 Cottman Ave., Philadelphia, 19111, PA

来源：

Moscow University Biological Sciences Bulletin | 2016年 / 71卷 / 2期

关键词：

chromatin; fluorescence microscopy; immobilization; nucleosome; single molecule;

D O I：

10.3103/S0096392516020048

中图分类号：

学科分类号：

摘要：

An experimental setup for study of immobilized molecules and their complexes by fluorescence microscopy with sensitivity at a single fluorophore level was developed. The installation records fluorescence images of immobilized molecules in two spectral ranges simultaneously allowing analysis based on the Förster resonance energy transfer effect. The fluorescence excitation is caused by evanescent light wave formed by the total internal reflection effect. Registration of signal is conducted with a highly sensitive detection system that allows measurements with a temporal resolution of approximately 100 ms. Glass surface modification protocol was developed for immobilization of nucleosomes via high-affinity streptavidin-biotin interactions. To ensure immobilization, one of the ends of the fluorescently labeled nucleosomal DNA was biotinylated. The algorithm of image processing for analysis of structural rearrangements at a single nucleosome level was developed. Fluorescence microscopy of single immobilized molecules and their complexes allows the analysis of nucleosome structural dynamics during transcription, and its interactions with various nuclear proteins. © 2016, Allerton Press, Inc.

引用

页码：97 / 101

页数：4

共 8 条

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