Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions

Abstract

Source: Gijsbers, A., et al. Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions. J. Vis. Exp. (2016).

In this video, we describe the fluorescence anisotropy technique to study the interactions between the fluorophore-tagged Shwachman-diamond syndrome (SBDS) protein and the elongation factor-like 1 GTPase (EFL1). On incubating SBDS proteins with gradually increasing concentrations of EFL1, a steady increase in anisotropy is observed, indicating a successful interaction between the two proteins.

Protocol

1. Labeling of SBDS-FlAsH with the FlAsH Fluorescent Dye 4',5'-Bis(1,3,2 dithioarsolan-2-yl) Fluorescein

1. Mix 3 nmol of the SBDS-FlAsH protein (the FlAsH-tag corresponds to the tetracysteine motif Cys-Cys-Pro-Gly-Cys-Cys genetically engineered in the C-terminus of the recombinant SBDS protein) with 3 nmol of the 4',5'-bis(1,3,2 dithioarsolan-2-yl) fluorescein dye in 5 µl volume of Anisotropy buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM MgCl₂, 10% glycerol, 5 mM β-mercaptoethanol).
2. Let the reaction proceed for 8 hr at 4 °C. Dialyze the sample against Anisotropy buffer overnight to remove the free dye.
3. Use th....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tris Base	Formedium	TRIS01	Ultra pure
dye 4&rsquo;,5&rsquo;-bis(1,3,2 dithioarsolan-2- yl) fluorescein	ThermoFischer Scientific	LC6090	This kit contains the dye to label a<br/> FlAsH tag
Fluorescence cell&nbsp;	Hellma Analytics&nbsp;	111-057-40
Spectrophotometer&nbsp;	Agilent Technologies&nbsp;	G6860AA&nbsp;	Cary 60 UV-Vis
Spectrofluorometer	Olis	No applicable	Olis DM 45 with Polarization Toolbox
2-Mercaptoethanol	Sigma-Aldrich	M6250-100ML
NaCl	Formedium	NAC02	Sodium Chloride
MgCl<sub>2</sub>	Merck Millipore Corporation	1725711000	Magnesium Chloride
Glycerol&nbsp;	Sigma-Aldrich&nbsp;	G5516
Glycerol	Tecsiquim, S.A. de C.V.	GT1980-6