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Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy
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Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy
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Journal Article
Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy
2012
Overview
High-speed atomic force microscopy (HS-AFM) allows direct visualization of dynamic structural changes and processes of functioning biological molecules in physiological solutions, at subsecond to sub-100-ms temporal and submolecular spatial resolution. Unlike fluorescence microscopy, wherein the subset of molecular events that you see is dependent on the site where the probe is placed, dynamic molecular events unselectively appear in detail in an AFM movie, facilitating our understanding of how biological molecules function. Here we present protocols for HS-AFM imaging of proteins in action, including preparation of cantilever tips, step-by-step procedures for HS-AFM imaging, and recycling of cantilevers and sample stages, together with precautions and troubleshooting advice for successful imaging. The protocols are adaptable in general for imaging many proteins and protein–nucleic acid complexes, and examples are described for looking at walking myosin, ATP-hydrolyzing rotorless F
1
-ATPase and cellulose-hydrolyzing cellulase. The entire protocol takes 10–15 h, depending mainly on the substrate surface to be used.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ ATP
/ Computational Biology/Bioinformatics
/ Imaging
/ Methods
/ Microscopy, Atomic Force - instrumentation
/ Microscopy, Atomic Force - methods
/ Myosin
/ Proteins
/ Protocol
/ Proton-Translocating ATPases - analysis
/ Proton-Translocating ATPases - chemistry
/ Proton-Translocating ATPases - metabolism
/ Scanners
