Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

A molecular stabiliser of an inhibitory eIF2B-eIF2(αP) complex activates the Integrated Stress Response.

Nature communications·2026

Same author

Metabolite import by SLC33A1 is required for ATF6 activation during endoplasmic reticulum stress.

Life science alliance·2026

Same author

Drug-related pressure ulcers in hospitalised patients: a multicentre retrospective study in Japan identifying high-risk medications and patient characteristics.

BMJ open·2025

Same author

A structural basis for chaperone repression of stress signaling from the endoplasmic reticulum.

Molecular cell·2025

Same author

Translon: a single term for translated regions.

Nature methods·2025

Same author

Inhibition of virally induced TFEB proteasomal degradation as a host-centric therapeutic approach for coronaviral infection.

Science advances·2025

Search research articles

Related Experiment Video

Updated: Oct 3, 2025

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Published on: May 1, 2020

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

Yusuke Sekine¹, David Ron², Alisa F Zyryanova²

¹Division of Endocrinology and Metabolism, Department of Medicine, Aging Institute, University of Pittsburgh, Pittsburgh, PA, USA. SEKINEY@pitt.edu.

Methods in Molecular Biology (Clifton, N.J.)

|February 16, 2022

Summary

This study presents a new in vitro method to measure the guanine nucleotide-exchange factor (GEF) activity of eukaryotic translation initiation factor 2B (eIF2B) toward its substrate eIF2. The protocol uses fluorescent GDP to quantify GEF activity in cell extracts.

Keywords:

Guanine nucleotide exchange factor (GEF)Guanine nucleotide-binding protein (G-protein)Integrated stress response (ISR)Translation Translation initiation factor eIF2 eIF2B

More Related Videos

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Published on: June 3, 2021

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Published on: November 15, 2013

Related Experiment Videos

Last Updated: Oct 3, 2025

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Published on: May 1, 2020

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Published on: June 3, 2021

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Published on: November 15, 2013

Area of Science:

Molecular biology
Biochemistry
Cellular biology

Background:

Guanine nucleotide-exchange factors (GEFs) are crucial regulators of G-protein signaling.
eIF2B is a key GEF involved in the regulation of eukaryotic translation initiation.
Accurate measurement of GEF activity is essential for understanding cellular signaling pathways.

Purpose of the Study:

To develop and describe a protocol for measuring the in vitro GEF activity of eIF2B towards eIF2.
To provide a method for assessing eIF2B GEF activity in crude cell extracts.
To enable kinetic analysis of the GEF reaction.

Main Methods:

Preparation of the eIF2 heterotrimeric substrate (phosphorylated or unphosphorylated eIF2α) via immunoprecipitation.
Loading of eIF2 with a fluorescent BODIPY-FL dye-attached GDP.
Kinetic monitoring of the exchange of fluorescent GDP for unlabeled GDP on eIF2, catalyzed by eIF2B, using a fluorescence microplate reader.

Main Results:

The described protocol allows for the measurement of eIF2B GEF activity in vitro.
The method is applicable to crude cell extracts, simplifying sample preparation.
Kinetic analysis of the GDP/GTP exchange reaction is feasible with this approach.

Conclusions:

This protocol offers a straightforward method for quantifying eIF2B GEF activity.
The assay facilitates the study of factors influencing eIF2B function in translation regulation.
This technique can be valuable for research in molecular biology and cell signaling.