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
  • 1. Division of Endocrinology and Metabolism, Department of Medicine, Aging Institute, University of Pittsburgh, Pittsburgh, PA, USA. [email protected].
  • 2. Cambridge Institute for Medical Research (CIMR), University of Cambridge, Cambridge, UK.
PMID: 35171481 DOI: 10.1007/978-1-0716-1975-9_12
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.