The Dcaf11-driven p21 ubiquitination assay kit is a sensitive AlphaLisa® high-throughput screening (HTS) assay kit, designed to measure Dcaf11 (DDB1- and Cul4-associated factor 11) E3 ligase activity in a homogeneous 384 reaction format. The assay kit comes with enough biotinylated ubiquitin, ATP, GST-tagged p21, assay buffer, detection buffer, purified UBE1 (E1), UbcH5b (E2), and Rbx1/Cul4B/Dcaf11/Ddb1 complex (E3) for 384 reactions. The assay detects mono-ubiquitination and poly-ubiquitination of p21.

Figure 1. Dcaf11-driven p21 ubiquitination assay kit schematic.
First, E1 and E2 enzymes are incubated with the E3 complex and with the target protein p21 (GST-tag) in the presence of biotin-conjugated ubiquitin and ATP. Ubiquitination of p21 occurs in a multistep ubiquitin transfer from E1 to E2 to E3, and E3-mediated conjugation of ubiquitin to p21. Next, glutathione-acceptor beads are added, followed by streptavidin-conjugated donor beads, and Alpha-counts are measured. The increase in Alpha-counts is proportional to the mono- or poly-ubiquitination of the GST-tagged p21.