Fluorescence Anisotropy to Determine Transcription Factor-DNA Binding Affinity

Published: June 29, 2023

Abstract

Source: Jung, C. et al., High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy. J. Vis. Exp. (2019).

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…

Declarações

The authors have nothing to disclose.

Materials

Cy5-labled 16- / 18-bp DNA-oligomers Eurofins Custom synthesis
16- / 18-bp DNA-oligomers Eurofins Custom synthesis
Nile Blue A Sigma N5632-25G
Sensoplate plus microplate 96- or 384-well, PS Greiner 655891
384-Well Sensoplate, black Greiner 788896
Agarose, low gelling temperature Sigma A9414-50G
Sodium Chloride  Merck  1.06404.1000
Tween-20  Sigma  P1379-1L
Di-Potassium hydrogen phosphate trihydrate Merck  1.05099.1000
Potassium dihydrogen phosphate Merck 1.04873.1000
Q-POD Element Merck Millipore ZMQSP0DE1
Millipak 40 Gamma Gold Filter Merck Millipore MPGL04GK2
Milli-Q Integral 3 Water Purification System Merck Millipore ZRXQ003WW
Quantum TIX Merck Millipore QTUMOTIX1
DL-Dithiothreitol Sigma 43815-1G
Mastercycler gradient Eppendorf Z316083
SafeSeal tube 1.5 mL Sarstedt 72.706.200
Tube 15 mL Sarstedt 62.554.502
Multiply-Pro cup 0.2 mL PP Sarstedt 72.737.002
MICROSCOPY SETUP:
Automated widefield microscope LEICA DMI6000
Long distance objective LEICA HCX PL FLUOAR L 60x/0.60 N.A. Dry
638 nm line continuous diode laser Omicron PHOxX 638-40, 40mW
Back-illuminated EM-CCD Camera Andor iXon DV897
Dichroic mirror AHF 640nm cut-off
Bandpass filter AHF ET bandpass 700/75
Linear polarizer Thorlabs LPVISC050-MP2
Polarizing beam splitter Thorlabs BS010
Achromatic lens Thorlabs 200 mm focal length
Multimode optical fiber Optronis FVP600660710
ROBOTIC SYSTEM:
Our robotic system includes a Biomek NXP workstation with a 96-channel head and Span-8 pipettors, connected with a servo-shuttle, which are used for all liquid transfer steps. In addition, the system is equipped with orbital shakers and a microplate reader (Paradigm, Molecular device) served by the Span-8 gripper Beckman Coulter Biomek NXP
SOFTWARE:
Programming language National Instruments Labview 9.0
Script for the HiP-FA software available at https://github.com/ GeneCenterMunich/HiP-FA

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Fluorescence Anisotropy to Determine Transcription Factor-DNA Binding Affinity. J. Vis. Exp. (Pending Publication), e21434, doi: (2023).

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