Fluorescence Intensity-Based eIF2B's Guanine Nucleotide-Exchange Factor Activity Assay.
Methods Mol Biol
; 2428: 187-196, 2022.
Article
em En
| MEDLINE
| ID: mdl-35171481
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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fator de Iniciação 2 em Eucariotos
/
Fatores de Troca do Nucleotídeo Guanina
Idioma:
En
Revista:
Methods Mol Biol
Ano de publicação:
2022
Tipo de documento:
Article