Rad23 UBL mutants show no detectable interaction with the proteasome in vitro. All Rad23 constructs were bacterially expressed, purified and radiolabelled with [γ-32P]ATP in vitro via an N-terminal HMK site. After quantification by scintillation counting and by Bradford assay, the radiolabelled proteins were normalized by counts and mixed with purified yeast proteasomes in a 1:10, 1:20 and 1:50 molar excess of proteasome over proteins. After incubation at 30°C for 15 minutes, the mixtures were electrophoresed on a 3.5% native gel at 100 V for 5 hours at 4°C. The arrows indicate free Rad23 proteins and the triangles indicate bands corresponding to proteasome-bound Rad23 proteins. "I/P" denotes the lane containing the input radiolabelled protein without any proteasome.