conformational changes we have studied.

Conclusions
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The tethered particle motion method is one of the simplest tools for performing single-molecule experiments on DNA-protein complexes. In contrast to other methods involving fluorescence, TPM never bleaches, allowing very long observations. The central idea is to use the Brownian motion of a small particle tethered to a DNA molecule as a reporter of the underlying macromolecular dynamics of the DNA in its complexes with DNA-binding proteins. The point of this paper has been to examine the challenges that are inherent in making useful quantitative measurements using this method. One of the main outcomes of that effort has been the development of calibration curves that illustrate how tethered-particle excursions depend upon both bead size and tether length.

Materials and Methods
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Sample preparation
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The first step in any TPM experiment is construction of the relevant DNA tethers with their associated reporter beads. Polymerase Chain Reaction (PCR) was used to amplify labeled DNA with two modified primers. The primers were either biotin or digoxigenin labeled at the 5’ ends (MWG Biotech AG, Ebersberg, Germany). The labels permit specific linkage of the DNA to a polystyrene microsphere or glass coverslip, respectively. The PCR templates 