| Literature DB >> 35171481 |
Yusuke Sekine1, David Ron2, Alisa F Zyryanova2.
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.Entities:
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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Year: 2022 PMID： 35171481 DOI： 10.1007/978-1-0716-1975-9_12
Source DB: PubMed Journal: Methods Mol Biol ISSN： 1064-3745