1. Academic Validation
  2. Fluorescence Intensity-Based eIF2B's Guanine Nucleotide-Exchange Factor Activity Assay

Fluorescence Intensity-Based eIF2B's Guanine Nucleotide-Exchange Factor Activity Assay

  • Methods Mol Biol. 2022;2428:187-196. doi: 10.1007/978-1-0716-1975-9_12.
Yusuke Sekine 1 David Ron 2 Alisa F Zyryanova 2
Affiliations

Affiliations

  • 1 Division of Endocrinology and Metabolism, Department of Medicine, Aging Institute, University of Pittsburgh, Pittsburgh, PA, USA. SEKINEY@pitt.edu.
  • 2 Cambridge Institute for Medical Research (CIMR), University of Cambridge, Cambridge, UK.
Abstract

Guanine nucleotide-exchange factors (GEFs) activate the function of guanine nucleotide-binding proteins (G-proteins) by promoting the exchange of GDP for GTP on the latter. Here, we describe a protocol for in vitro measurements of the GEF activity of eukaryotic translation initiation factor 2B, eIF2B, toward its substrate eIF2. This protocol provides a relatively simple method for determining the eIF2B's GEF activity in crude cell extracts. The eIF2 heterotrimeric substrate, with phosphorylated or unphosphorylated eIF2α, is prepared by immunoprecipitation, following subsequent loading of a fluorescent BODIPY-FL dye-attached GDP. The exchange of the bound fluorescent GDP molecule for an unlabeled one on eIF2 promoted by eIF2B is monitored kinetically using a fluorescence microplate reader.

Keywords

Guanine nucleotide exchange factor (GEF); Guanine nucleotide-binding protein (G-protein); Integrated stress response (ISR); Translation; Translation initiation factor; eIF2; eIF2B.

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