Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Among the eight possible linkages, K29-linked polyubiquitin is a relatively abundant type of polyubiquitin in both human and yeast cells. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. We screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation.