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

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.