Cell Signaling Technology

Product Pathways - Protein Stability

NEDD8 Blocking Peptide #1048

Description

This peptide is used to block NEDD8 (19E3) Rabbit mAb #2754 reactivity.

Quality Control

The quality of the peptide was evaluated by reversed-phase HPLC and by mass spectrometry. The peptide blocks NEDD8 (19E3) Rabbit mAb #2754 by immunohistochemistry.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using NEDD8 (19E3) Rabbit mAb #2754 in the presence of control peptide (left) or NEDD8 Blocking Peptide (right).

Applications

Use as a blocking reagent to evaluate the specificity of antibody reactivity in immunohistochemistry protocols.

Directions for Use

For immunohistochemistry, add twice the volume of peptide as volume of antibody used in 100 μl total volume. Incubate for a minimum of 30 minutes prior to adding the entire volume to the slide. Recommended antibody dilutions can be found on the product data sheet.

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

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Companion Products

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

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