Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions

Published: May 31, 2023

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 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-mercap…

Disclosures

The authors have nothing to disclose.

Materials

Tris Base Formedium TRIS01 Ultra pure
dye 4’,5’-bis(1,3,2 dithioarsolan-2- yl) fluorescein ThermoFischer Scientific LC6090 This kit contains the dye to label a
FlAsH tag
Fluorescence cell  Hellma Analytics  111-057-40
Spectrophotometer  Agilent Technologies  G6860AA  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
MgCl2 Merck Millipore Corporation 1725711000 Magnesium Chloride
Glycerol  Sigma-Aldrich  G5516
Glycerol Tecsiquim, S.A. de C.V. GT1980-6

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Cite This Article
Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions. J. Vis. Exp. (Pending Publication), e21411, doi: (2023).

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