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Covalently Linked Protein Regulators

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.

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Related Experiment Video

Updated: Jul 5, 2026

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Published on: January 29, 2018

Analysis of protein sumoylation.

Roland S Hilgarth¹, Kevin D Sarge¹

¹University of Kentucky, Lexington, Kentucky.

Current Protocols in Protein Science

|April 23, 2008

Summary

Identifying new sumoylated proteins and their modification sites is crucial for understanding cellular regulation. This study presents two methods, immunoprecipitation and in vitro reactions, to detect protein sumoylation and map SUMO attachment sites.

Area of Science:

Biochemistry
Molecular Biology
Cellular Biology

Background:

Sumoylation, the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to target proteins, is a critical post-translational modification regulating diverse cellular processes.
Identifying novel sumoylated proteins and the specific lysine residues involved is essential for elucidating the functional outcomes of this modification.

Purpose of the Study:

To describe experimental approaches for identifying specific protein sumoylation.
To enable the determination of functional consequences of sumoylation through targeted mutagenesis.

Main Methods:

Immunoprecipitation of the protein of interest followed by small ubiquitin-like modifier (SUMO) immunoblotting.
In vitro sumoylation enzymatic reaction using in vitro translated or purified recombinant proteins, followed by SDS-PAGE analysis to detect higher molecular-weight bands indicating sumoylation.

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Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

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Last Updated: Jul 5, 2026

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Published on: January 29, 2018

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Published on: November 2, 2017

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Published on: May 3, 2015

Main Results:

The described methods allow for the identification of sumoylated proteins.
Specific lysine residues targeted for SUMO attachment can be determined, facilitating further functional studies.

Conclusions:

Two distinct experimental strategies are provided for robust detection and characterization of protein sumoylation.
These methods are valuable tools for researchers investigating the role of sumoylation in cellular regulation and disease.