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Fluorescence Anisotropy to Determine Transcription Factor-DNA Binding Affinity
Overview
This video describes high-performance fluorescence anisotropy that helps to monitor the interaction between transcription factors and DNA. The assay monitors the interactions of a fluorophore-labeled DNA molecule with its specific transcription factor by measuring the degree of polarization due to molecular rotation or anisotropy of the fluorophore-labeled DNA.
Protocol
1. Polarization Microscopy
- For widefield laser illumination, focus a 638 nm line of a continuous diode laser (40 mW) on the aperture of multimode optical fiber for beam cleanup. Mount a linear polarizer at the output of the fiber to set the polarization of laser light.
- Block the excitation component of the emitted light with a dichroic mirror (640 nm cut-off) and a bandpass filter (bandpass 700/75).
- Let the fluorescence signal pass through a polarizing beam splitter, whic.......
Representative Results
Figure 1: Schematic depictions of the HIP-FA assay and experimental setup. (a) Gel delivery system for titrating competitor DNA in single wells. (b) HIP-FA microscopy setup.Customized automated widefield microscope with polarized fluorescence light detection on an EM-CCD camera. (c) Raw fluorescence image with the two regions of interest used to determine the parallel (red) and the perpendic.......
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Source: Jung, C. et al., High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy. J. Vis. Exp. (2019).
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