Figure 1 |.

Overview of the ligase trapping procedure. F-box-UBA domain fusion proteins (i.e., ligase traps) are expressed in cells at physiological levels along with overexpression of a single copy of the ubiquitin gene containing an N-terminal hexahistidine epitope tag. The UBA of the ligase trap interacts with the nascent ubiquitin chain on endogenous SCF substrates, thereby delaying their release (left). Cells are then lysed and subjected to an anti-FLAG coimmunoprecipitation under native conditions to isolate ligase trap complexes (center). FLAG eluates are collected and a second purification is performed using Ni-NTA agarose beads under denaturing conditions to capture ubiquitinated substrates (right). This second step eliminates interactors associated with the ubiquitinated species that are not themselves substrates. FL, FLAG; H, histidine.