49 Result(s)
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Protocol
Single-Molecule FRET-Resolved Protein Dynamics – from Plasmid to Data in Six Steps
Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for the detection of conformational dynamics of biomolecules. While many smFRET experiments are performed using dye-labeled DN...
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Protocol
Probing Mitotic Chromosome Mechanics Using Optical Tweezers
During mitosis, cells compact their DNA into rodlike shapes, four orders of magnitude shorter than the DNA backbone contour length. We describe an experimental protocol to isolate and study these intricate mit...
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Protocol
Atomic Force Microscopy of Viruses: Stability, Disassembly, and Genome Release
In atomic force microscopy (AFM), the probe is a nanometric tip located at the end of a microcantilever which palpates the specimen under study as a blind person manages a walking stick. In this way, AFM allow...
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Protocol
Single-Molecule Fluorescence Microscopy in Sensory Cilia of Living Caenorhabditis elegans
Intracellular transport of organelles and biomolecules is vital for several cellular processes. Single-molecule fluorescence microscopy can illuminate molecular aspects of the dynamics of individual biomolecul...
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Protocol
Visualizing Molecular Dynamics by High-Speed Atomic Force Microscopy
Dynamic processes and structural changes of biological molecules are essential to life. While conventional atomic force microscopy (AFM) is able to visualize molecules and supramolecular assemblies at sub-nano...
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Protocol
Detecting DNA Loops Using Tethered Particle Motion
The range of motion of a micron-sized bead tethered by a single polymer provides a dynamic readout of the effective length of the polymer. The excursions of the bead may reflect the intrinsic flexibility and/o...
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Protocol
Fluorescence Microscopy of Nanochannel-Confined DNA
Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level, and the polymer physics of confined DNA and ...
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Protocol
Surface Functionalization, Nucleic Acid Tether Characterization, and Force Calibration for a Magnetic Tweezers Assay
Magnetic tweezers are a force spectroscopy single-molecule technique. They enable the mechanical manipulation of biomolecules via the means of a magnetic particle under an attractive force applied by a magneti...
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Protocol
DNA Origami-Based Single-Molecule Force Spectroscopy and Applications
Over the last years, single-molecule force spectroscopy provided insights into the intricate connection between mechanical stimuli and biochemical signaling. The underlying molecular mechanisms were uncovered ...
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Protocol
Single-Molecule FRET X
Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon that has been repurposed as a biophysical tool to measure nanometer distances. With FRET by DNA eXchange, or FRET X, many points of i...
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Protocol
Quantifying ATP-Independent Nucleosome Chaperone Activity with Single-Molecule Methods
The dynamics of histone-DNA interactions govern chromosome organization and regulates the processes of transcription, replication, and repair. Accurate measurements of the energies and the kinetics of DNA bind...
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Protocol
Measuring Transcription Dynamics of Individual Genes Inside Living Cells
Transcription is a highly dynamic process, which, for many genes, occurs in stochastic bursts. Studying what regulates these stochastic bursts requires visualization and quantification of transcription dynamic...
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Protocol
Insect Cell-Based Expression of Cytoskeletal Motor Proteins for Single-Molecule Studies
Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and kinesin superfamilies play critical roles in...
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Protocol
Atomic Force Microscopy: An Introduction
Imaging of nano-sized particles and sample features is crucial in a variety of research fields, for instance, in biological sciences, where it is paramount to investigate structures at the single particle leve...
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Protocol
A Brief Introduction to Single-Molecule Fluorescence Methods
One of the most popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods prov...
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Protocol
Unfolding and Refolding Proteins Using Single-Molecule AFM
Single-molecule atomic force microscopy (AFM) allows capturing the conformational dynamics of an individual molecule under force. In this chapter, we describe a protocol for conducting a protein nanomechanical...
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Protocol
Correlated Single-Molecule Magnetic Tweezers and Fluorescence Measurements of DNA-Enzyme Interactions
Combining force spectroscopy and fluorescence microscopy provides a substantial improvement to the single-molecule toolbox by allowing simultaneous manipulation and orthogonal characterizations of the conforma...
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Protocol
Lattice Light-Sheet Motor-PAINT: A Method to Map the Orientations of Microtubules in Complex Three-Dimensional Arrays
Microtubules play an essential role in many cellular functions, in part by serving as tracks for intracellular transport by kinesin and dynein. The ability of microtubules to fulfill this role fundamentally de...
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Protocol
An Introduction to Magnetic Tweezers
Magnetic tweezers are a single-molecule force and torque spectroscopy technique that enable the mechanical interrogation in vitro of biomolecules, such as nucleic acids and proteins. They use a magnetic field ...
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Protocol
Single-Cell Measurements Using Acoustic Force Spectroscopy (AFS)
Single-molecule force spectroscopy is a powerful tool to investigate the forces and motions related to interactions of biological molecules. Acoustic force spectroscopy (AFS) is a developed measurement tool to...
