Detection and analysis of protein ISGylation

Tomoharu Takeuchi; Hideyoshi Yokosawa

doi:10.1007/978-1-60327-084-7_10

Detection and analysis of protein ISGylation

Methods Mol Biol. 2008:446:139-49. doi: 10.1007/978-1-60327-084-7_10.

Authors

Tomoharu Takeuchi¹, Hideyoshi Yokosawa

Affiliation

¹ Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.

PMID: 18373255
DOI: 10.1007/978-1-60327-084-7_10

Abstract

ISG15 is a ubiquitin-like modifi er that is conjugated to target proteins by a sequential reaction catalyzed by E1/E2/E3 enzymes (protein ISGylation). ISG15 and protein ISGylation are upregulated by interferon stimuli. ISG15 functions as an antiviral protein against Sindbis virus and HIV-1, but the molecular mechanism remains unknown. Here we describe in detail methods for detecting and analyzing protein ISGylation. The methods consist of plasmid transfection and affi nity purifi cation of ISGylated proteins. In addition, we describe a method for detecting ISGylation of a target protein, Ubc13.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Blotting, Western
Chromatography, Affinity
Cytokines / metabolism*
Electrophoresis, Polyacrylamide Gel
Immunoprecipitation
Proteins / metabolism*
Transfection
Ubiquitins / metabolism*

Substances

Cytokines
Proteins
Ubiquitins
ISG15 protein, human