A fluorescence polarization-based competition assay for measuring interactions between unlabeled ubiquitin chains and UCH37•RPN13.

ABSTRACT: Ubiquitin chains regulate distinct signaling events through cooperative interactions with effector proteins and deubiquitinases. Measuring the strength of these interactions is often challenging; either large amounts of material are required or one of the binding partners must be labeled for detection. We sought to develop a label-free method for measuring binding of ubiquitin chains to the proteasome-associated deubiquitinase UCH37 and its binding partner RPN13. The method we describe here is based on a fluorescence polarization competition (FP_comp) assay in which fluorescent monoubiquitin is competed off the UCH37•RPN13 complex by the addition of unlabeled ubiquitin chains. We show that the UCH37•RPN13 complex displays higher affinity toward chains with more than two ubiquitin

SUBMITTER: Du J 

PROVIDER: S-EPMC5976553 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature