Imaging of DNA and protein by SFM and combined SFM-TIRF microscopy

Małgorzata Grosbart, Dejan Ristić, Humberto Sánchez, Claire Wyman

Research output: Chapter in Book/Conference proceedings/Edited volumeChapterScientificpeer-review

3 Citations (Scopus)

Abstract

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nm resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherSpringer
Pages259-280
Number of pages22
Volume1665
ISBN (Electronic)978-1-4939-7271-5
ISBN (Print)978-1-4939-7270-8
DOIs
Publication statusPublished - 2018

Publication series

NameMethods in Molecular Biology
Volume1665
ISSN (Print)1064-3745

Keywords

  • Atomic force microscopy
  • Combining fluorescence and topography
  • DNA-protein complexes
  • Scanning force microscopy
  • Single-molecule imaging

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