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

Yusuke Sekine, David Ron, Alisa F Zyryanova

Methods in molecular biology (Clifton, N.J.) 2022

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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. © 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Yusuke Sekine, David Ron, Alisa F Zyryanova. Fluorescence Intensity-Based eIF2B's Guanine Nucleotide-Exchange Factor Activity Assay. Methods in molecular biology (Clifton, N.J.). 2022;2428:187-196