Fluorescence Anisotropy to Determine Transcription Factor-DNA Binding Affinity

Samenvatting

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

1. 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.
2. Block the excitation component of the emitted light with a dichroic mirror (640 nm cut-off) and a bandpass filter (bandpass 700/75).
3. Let the fluorescence signal pass through a polarizing beam splitter, which splits the emitted light into its perpendicular and parallel polarized components. Then, focus the non-reflected beam (parallel component) and the r....

Resultaten

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.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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
<strong>MICROSCOPY SETUP:</strong>
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
<strong>ROBOTIC SYSTEM:</strong>
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
<strong>SOFTWARE:</strong>
Programming language	National Instruments	Labview 9.0
Script for the HiP-FA software available at			https://github.com/ GeneCenterMunich/HiP-FA