Ataxin-3 protein belongs to a novel group of cysteine proteases similar to USP-type ubiquitin proteases and has deubiquitinating activity in vitro. The full-length protein contains an catalytic N-terminal Josephin domain, three ubiquitin interacting motifs (UIMs), and a variable C-terminus with a polyglutamine stretch. Ataxin3 has deconjugating activity and functions as a mixed linkage, chain editing enzyme with preferential cleavage of K63 linkages in mixed chains. Ataxin3 also bind both K48-linked and K63-linked poly-Ub chains via its UIM domains and preferentially interacts with four or more ubiquitin units. This protein can be used for the isolation and identification of K48-linked (preferentially) or K63-linked poly-Ub chains or ubiquitinated substrates that contain these linkages. Detection with avidin-linked reagents allows for a higher efficiency and detection sensitivity than with other antibodies.