Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Ubiquitin is first activated by forming a thiolester complex with the activation component E1. The activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, and then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue. Research studies suggest that activated E2 associates transiently with E3, and the dissociation is a critical step for ubiquitination. S phase kinase associated protein 1 (Skp1) is a critical scaffold protein of the Skp1/CUL1/F-box (SCF) E3 ubiquitin ligase protein complex. Various F-box proteins (e.g. beta-TrCP, Skp2) mediate an interaction with Skp1 via their defining and conserved domain of 40aa and with substrates to be ubiquitinated. RING-box protein 1 (RBX1 or ROC1) is another essential component of the SCF complex. RBX1 mediates the neddylation of CUL1, which activates SCF E3 ligase by facilitating the ubiquitin transfer from E2 to substrates. The RING finger domain of RBX1 is required for ubiquitinligation. Cullin-4 (CUL4) is a member of the cullin family of related ubiquitin ligases. The carboxy-terminal domain of CUL4 interacts with Rbx1 and E2 enzyme while the amino-terminal CUL4 domain interacts with BPB domain of UV damaged DNA binding protein DDB-1 to form a CUL4-DDB1 ubiquitin ligase complex. Damaged DNA-Binding Protein(DDB) consists of a 127kD subunit (DDB-1) and a 48kD subunit (DDB-2) that contribute to the formation of the UV damaged DNA-binding protein complex (UV-DDB). In conjunction with CUL4A and RBX1, the UV-DDB complex forms an E3 ubiquitin ligase that recognizes a broad spectrum of DNA lesions. The complex polyubiquitinates components of the nucleotide excision repair pathway.
Specificity:Human
Intended Use:The Ubiquitin E3 Ligase Complex Antibody Sampler BioAssay™ Kit provides an economical means to study multiple protein components of ubiquitin E3 ligase complexes. The kit includes enough antibody to perform two western blot experiments per primary antibody.
Applications:Suitable for use in Western Blot. Other applications not tested.
Recommended Dilution:Western Blot: 1:500-1:2000Optimal dilutions to be determined by the researcher.
Storage and Stability:May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
