Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 931; p. 295
• Main Authors: Shlyakhtenko, Luda S, Gall, Alexander A, Lyubchenko, Yuri L
• Format: Journal Article
• Language: English
• Published: United States 01.01.2013
• Subjects: Aluminum Silicates - chemistry; Bridged Bicyclo Compounds, Heterocyclic - chemical synthesis; Bridged Bicyclo Compounds, Heterocyclic - chemistry; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - ultrastructure; Microscopy, Atomic Force - methods; Nucleosomes - chemistry; Nucleosomes - ultrastructure; Organosilicon Compounds - chemical synthesis; Organosilicon Compounds - chemistry; Plasmids - chemistry; Plasmids - ultrastructure; Propylamines; Silanes - chemistry; Surface Properties; Time-Lapse Imaging
• ISSN: 1940-6029
• DOI: 10.1007/978-1-62703-056-4_14

Surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes with atomic force microscopy (AFM). This article describes the approaches for chemical functionalization of the mica surface. One approach utilizes 3-aminopropyl-trietoxy silane (APTES), enabling one to obtain a smooth surface termed AP-mica. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations and in a broad range of pH. Another method utilizes aminopropyl silatrane (APS) to yield an APS-mica surface. The advantage of APS-mica compared with AP-mica is the ability to obtain reliable and reproducible time-lapse images in aqueous solutions. The chapter describes the methodologies for the preparation of AP-mica and APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The applications are illustrated with a number of examples.