  The Tethered Particle Motion (TPM) method has been used to observe and
characterize a variety of protein-DNA interactions including DNA looping 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 200bp to 2.6kbp
and consider particle diameters of 200, 490 and 970nm. We also present a
systematic comparison between measured particle excursions and theoretical
expectations, which helps clarify both the experiments and models of DNA
conformation.
