Cell Signaling Technology

Product Pathways - Protein Stability

NEDD8 Antibody #2745

Applications Reactivity Sensitivity MW (kDa) Source
W IP IHC-P F H M R Mk (X) (Z) (B) Endogenous 9 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  X=Xenopus  Z=Zebrafish  B=Bovine
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

This antibody detects endogenous levels of both free and conjugated NEDD8 protein. The antibody does not cross-react with other ubiquitin family members, including ubiquitin, SUMO1, SUMO2, SUMO3 and ISG15.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino acids at the amino-terminus of human NEDD8 protein. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of Ubiquitin, NEDD8, ISG15 and SUMO-2/3 recombinant proteins (5 ng each), using NEDD8, Ubiquitin, ISG15 and SUMO-2/3 Antibodies.

Western Blotting

Western Blotting

Western blot analysis of lysates from HeLa, Raji, RAW, C6 and COS cells, using NEDD8 antibody.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic and nuclear localization, using NEDD8 Antibody.


IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using NEDD8 Antibody in the presence of control peptide (left) or Nedd8 Blocking Peptide #1048 (right).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Raji cells, using NEDD8 Antibody (blue) compared to a nonspecific negative control antibody (red).

Background

Neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8), also known as Rub1 (related to ubiquitin 1) in plants and yeast, is a member of the ubiquitin-like protein family (1,2). The covalent attachment of NEDD8 to target proteins, termed neddylation, is a reversible, multi-step process analogous to ubiquitination. NEDD8 is first synthesized in a precursor form with a carboxy-terminal extension peptide that is removed by either the UCH-L3 or NEDP1/DEN1 hydrolase protein to yield a mature NEDD8 protein (3,4). Mature NEDD8 is then covalently linked to target proteins via the carboxy-terminal glycine residue in a reaction catalyzed by the APP-BP1/Uba3 heterodimer complex and Ubc12 as the E1- and E2-like enzymes, respectively (5). An E3 ligase protein, Roc1/Rbx1, is also required for neddylation of the cullin proteins (6). Protein de-neddylation is catalyzed by a number of enzymes in the cell, including a "ubiquitin-specific" protease USP21, the NEDP1/DEN1 hydrolase and the COP9/signalosome (CSN) (7,8,9). In contrast to the ubiquitin pathway, the NEDD8 modification system acts on only a few substrates and does not appear to target proteins for degradation. Neddylation of cullin proteins activates the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complex by promoting complex formation and enhancing the recruitment of the E2-ubiquitin intermediate (10). While NEDD8 modification of VHL is not required for ubiquitination of HIF1-α, it is required for fibronectin matrix assembly (11). Mdm2-dependent neddylation of p53 inhibits its transcriptional activity (12).

  1. Chiba, T. and Tanaka, K. (2004) Curr. Protein Pept. Sci. 5, 177-184.
  2. Schwartz, D.C. and Hochstrasser, M. (2003) Trends Biochem. Sci. 28, 321-328.
  3. Wada, H. et al. (1998) Biochem. Biophys. Res. Commun. 251, 688-692.
  4. Hemelaar, J. et al. (2004) Mol. Cell Biol. 24, 84-95.
  5. Osaka, F. et al. (1998) Genes Dev. 12, 2263-2268.
  6. Kamura, T. et al. (1999) Genes Dev. 13, 2928-2933.
  7. Gong, L. et al. (2000) J. Biol. Chem. 275, 14212-14216.
  8. Mendoza, H.M. et al. (2003) J. Biol. Chem. 278, 25637-25643.
  9. Lyapina, S. et al. (2001) Science 292, 1382-1385.
  10. Kawakami, T. et al. (2001) EMBO J. 20, 4003-4012.
  11. Stickle, N.H. et al. (2004) Mol. Cell Biol. 24, 3251-3261.
  12. Xirodimas, D.P. et al. (2004) Cell 118, 83-97.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

Products