Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

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

Ribonuclease RNase Z is an evolutionarily conserved deAMPylase.

Proceedings of the National Academy of Sciences of the United States of America·2025
Same author

A repurposed AMP binding domain reveals mitochondrial protein AMPylation as a regulator of cellular metabolism.

Nature communications·2025
Same author

The rise of AMPylation: from bacterial beginnings to modern implications in health and disease.

Biochemical Society transactions·2025
Same author

Selenoprotein O Promotes Melanoma Metastasis and Regulates Mitochondrial Complex II Activity.

Cancer research·2024
Same author

A murine model of hnRNPH2-related neurodevelopmental disorder reveals a mechanism for genetic compensation by Hnrnph1.

The Journal of clinical investigation·2024
Same author

A murine model of hnRNPH2-related neurodevelopmental disorder reveals a mechanism for genetic compensation by Hnrnph1.

The Journal of clinical investigation·2023

Related Experiment Video

Updated: Jun 15, 2025

Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays
03:09

Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays

Published on: August 9, 2024

606

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O.

Abner Gonzalez1, Anju Sreelatha2

  • 1Department of Physiology, University of Texas Southwestern Medical Center.

Journal of Visualized Experiments : Jove
|August 26, 2024
PubMed
Summary

This study introduces a thermal shift assay (TSA) to investigate protein function by measuring changes in melting temperature. This method efficiently tests metal and nucleotide binding for proteins like Selenoprotein O (SelO), aiding in understanding cellular processes.

More Related Videos

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors
08:45

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

Published on: July 17, 2020

6.2K
An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
09:58

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Published on: November 29, 2016

15.6K

Related Experiment Videos

Last Updated: Jun 15, 2025

Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays
03:09

Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays

Published on: August 9, 2024

606
Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors
08:45

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

Published on: July 17, 2020

6.2K
An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
09:58

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Published on: November 29, 2016

15.6K

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Understanding proteins with unknown functions is crucial for deciphering cellular processes.
  • In vitro studies of such proteins are often limited by the need for specific cofactors and optimal conditions.
  • Cofactor binding can influence both protein activity and thermal stability.

Purpose of the Study:

  • To present a protocol for Thermal Shift Assay (TSA) for high-throughput screening of ligand binding.
  • To utilize TSA to investigate the cofactor binding properties of the pseudokinase, Selenoprotein O (SelO).
  • To provide insights into the molecular interactions governing SelO function through thermal stability changes.

Main Methods:

  • Implementation of Thermal Shift Assay (TSA) to measure protein melting temperature shifts.
  • Testing of various metal and nucleotide ligands for binding to SelO.
  • Analysis of ATP binding in an inverted orientation by SelO, contrasting with canonical kinases.

Main Results:

  • Demonstrated TSA as a simple and high-throughput method for assessing ligand interactions.
  • Identified specific metal and nucleotide binding interactions for SelO.
  • Characterized the unique ATP binding mode of SelO, essential for protein AMPylation.

Conclusions:

  • TSA is an effective method for probing ligand binding and determining optimal conditions for protein studies.
  • The findings elucidate the molecular basis of SelO's function in protein AMPylation.
  • This approach facilitates the study of proteins with unknown functions and their associated cellular roles.