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…
Divulgations
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 |
Tags
Citer Cet Article
Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions. J. Vis. Exp. (Pending Publication), e21411, doi: (2023).