Title

Using In Vitro Ubiquitylation Assays to Estimate the Affinities of Ubiquitin-Conjugating Enzymes for Their Ubiquitin Ligase Partners

Document Type

Book Section

Publication Date

9-22-2018

Publication Title

The Ubiquitin Proteasome System

Publisher

Humana Press

Publisher Location

New York

Volume

1844

First page number:

39

Last page number:

58

Abstract

Ubiquitin ligases (E3s) function by binding to both a protein substrate and to ubiquitin-conjugating enzymes (E2s) bound to ubiquitin. E3s facilitate the transfer of ubiquitin from the E2 active site to an E3-bound substrate. Thus, the affinity of the interaction of an E2 with its E3 partner is of considerable interest. The purpose of this work is to (1) provide protocols for the purification of the human E2 Cdc34, as well as for some additional protein components needed for the assays described here whose purification protocols haven’t been described elsewhere in detail; (2) provide the researcher with critical information regarding the proper long-term storage of these enzymes to retain maximal activity; (3) provide a protocol to benchmark Cdc34 activity with previously described activity levels in the literature; and (4) provide a simple and rapid means of measuring E2 affinity for an E3.

Keywords

Affinity; Cdc34; In vitro ubiquitylation; Protein-protein interaction; Skp1-Cullin Fbox (SCF) ubiquitin ligase; UbcH5; Ube2R1/2; Ubiquitin ligase; Ubiquitin-conjugating enzyme

Disciplines

Chemistry

Language

English

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