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Calibration of Tethered Particle Motion Experiments

  • Lin Han
  • Bertrand H. Lui
  • Seth Blumberg
  • John F. Beausang
  • Philip C. Nelson
  • Rob PhillipsEmail author
Conference paper
Part of the The IMA Volumes in Mathematics and its Applications book series (IMA, volume 150)

Abstract

The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA loping and transcription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead’s random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better characterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200 bp to 2.6 kbp and consider particle diameters of 200, 490 and 970 nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation.

Key words

Tethered particle DNA Brownian motion calibration single molecule 

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Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Lin Han
    • 1
  • Bertrand H. Lui
    • 1
    • 2
  • Seth Blumberg
    • 1
    • 3
  • John F. Beausang
    • 4
  • Philip C. Nelson
    • 4
  • Rob Phillips
    • 1
    Email author
  1. 1.Department of Applied PhysicsCalifornia Institute of TechnologyPasadenaUSA
  2. 2.Department of BioengineeringStanford UniversityStanfordUSA
  3. 3.University of Michigan Medical Scientist Training ProgramAnn ArborUSA
  4. 4.Department of Physics and AstronomyUniversity of PennsylvaniaPhiladelphiaUSA

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