The Circular Dichroism Spectroscopy Technique to Study DNA-Protein Interactions

Abstract

Source: Arya, V., et al. CD Spectroscopy to Study DNA-Protein Interactions. J. Vis. Exp. (2022).

This video demonstrates the circular dichroism (CD) spectroscopy technique to study conformational changes occurring in DNA in the presence of ATP-dependent chromatin remodeling protein. The changes in the CD spectra measured in the presence and absence of ATP hydrolysis indicate the importance of ATP in the ability of the remodeler to induce conformational changes in the bound DNA.

Protocol

1. Working concentration of the reaction components

1. Prepare the working concentrations of buffers for CD freshly and keep them at 4 °C before setting up the reactions.
   NOTE: For the CD reactions described in this paper, the working concentrations of components are as follows: Sodium phosphate buffer (pH 7.0) 1 mM, ATP 2 mM, DNA 500 nM, Protein 1 µM, MgCl₂ 10 mM, EDTA 50 mM, ADAADiN 5 µM.

2. Choosing and preparation of CD cuvettes

1. Collect CD spectra in high-transparency quartz cuvettes. Use rectangular or cylindrical cuvettes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenosine 5′-triphosphate disodium salt hydrate	Sigmaaldrich	A2383
CD Quartz Cuvette	STARNA	21-Q-1
Chirascan V100 CD spectrometer	Applied Photophysics	Not available
EDTA Disodium Salt Dihydrate	SRL	43272
Hellmanex III cleaning solution	Hellma	9-307-011-4-507
Magnesium Chloride Hexahydrate	Fisher scientific	M33-500
Sodium Phosphate Dibasic Anhydrous	Fisher scientific	S374-500
Sodium Phosphate Monobasic Monohydrate	Fisher scientific	S369-500
Synergy HT microplate reader	BioTek	Not available
Tris Base	Fisher scientific	BP152-500