High-throughput assay to monitor formation of the E2-ubiquitin thioester intermediate.
Targeting components of ubiquitination pathways for drug discovery necessitates the development of high-capacity assays that monitor the ubiquitination process at defined steps of the E1-E2-E3 cascade. This chapter describes the development of an assay based on time-resolved fluorescence to monitor formation of the thioester intermediate between ubiquitin-conjugating enzymes (E2s) and ubiquitin. The methodology is exemplified by an assay tailored for the ubiquitin-conjugating enzyme Cdc34. This assay setup can be easily adapted to other E2s and is suitable to screen small molecule inhibitors of E2-thioester formation in a high-throughput mode.
Hauser
Patrick
P
Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4002 Basel, Switzerland.
Hofmann
Francesco
F
D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D005063:Europium; D006801:Humans; D013194:Staining and Labeling; D013463:Sulfuric Acid Esters / Q000032:analysis* / Q000378:metabolism; D025801:Ubiquitin / Q000032:analysis* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000032:analysis* / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D013463:Sulfuric Acid Esters; D025801:Ubiquitin; D005063:Europium; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome