||Recombinant human CUL5 (amino acid residues 1-780), Rnf7 (amino acid residues 1-113) was expressed in Insect (Sf21) cells.
||Cullin-RING-Ligases (CRLs) are one largest class of ubiquitin E3 ligases and the enzymes of the NEDDylation pathway play a pivotal role in the activation of these, akin to ubiquitylation, the E1 activating enzyme (APP-BP1/UBA3 heterodimer) and the E2 conjugating enzymes (UBE2M or UBE2F) are involved in mammalian NEDDylation of the Cullin Ring Ligases (CRLs) (Meyer-Schaller et al., 2009; Huang et al., 2011; Morimoto et al., 2003).
||50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
||E3 Ligase Assay: The activity of Cul5/Rnf7 was validated indirectly through its ability to act as a substrate for Neddylation in the presence of the NEDD8 E3 ligase His-DCNL1 and thioester loaded His-Ube2M-NEDD8. Incubation of Cul5/Rnf7 and thioester loaded His-Ube2M-NEDD8 in the presence or absence of His-DCNL1 at 4?C was compared at two time points T0 and T2 minutes. Increased Neddylation of the Cul5 subunit in the presence of His-DCNL1 was demonstrated.
||12 months at -70°C. Avoid multiple freeze/thaw cycles.