Poly-ubiquitination of a target protein is a well-established mechanism to regulate proteasome mediated degradation; additionally, both poly- and mono-ubiquitination have been shown to control critical protein functions like protein: protein interaction, spatial regulation, and crosstalk with other post-translational modifications (PTMs). Because of its ubiquitous nature, and its clear importance in regulating an array of protein functions, it is no longer practical to leave the investigation of ubiquitination (Ub) to just a few ubiquitin experts. However, user-friendly tools are needed in order for non-Ub-specialists to efficiently study whether and under what conditions, their target protein is ubiquitinated. Here we describe a newly developed Signal-Seeker Ub kit (BK161) that rapidly identifies both poly- and mono-Ub for any target protein. To highlight the utility of this kit, detection of endogenous poly- and mono-Ub of Rac1 as well as chain specific ubiquitin detection was performed. In particular we compare the kits ubiquitin affinity beads, UBA01 with other commercially available ubiquitin affinity reagents. Collectively, these studies show that UBA01 is the most robust and precise mono- and poly-Ub detection reagent available, and utilization of the comprehensive BK161 kit will provide the best path to determining if a target protein is ubiquitinated.
