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