NEDDylation Kit



BML-UW0590-0001	1 Kit	304.00 USD

Generate NEDD8-E2 thioesters for use in the in vitro NEDDylation of target proteins.
Demonstrate novel proteins are potential targets for NEDDylation.
Study NEDD8 activation (E1) and conjugation (E2) steps in the NEDDylation cascade.
Generate substrates for deNEDDylating enzymes.
Test proteins for NEDD8 E3 ligase activity.

Product Specification

Quantity:	20 assays

Kit/Set Contains:	NEDD8 Activating Enzyme Solution (E1) UbcH12 Solution (E2) NEDD8 Solution NEDDylation Buffer Mg-ATP Solution NEDD8 Antibody Solution Non-reducing Gel Loading Buffer

Application:	This kit provides the means of generating a thioester-linked NEDD8-conjugated E2 enzyme, utilizing the first two steps in the NEDD8 cascade, for use in the NEDDylation of E3 ligases and target substrate proteins (user supplied). A NEDD8-specific antibody is provided for detection of NEDDylated proteins via SDS-PAGE and Western blotting. This kit provides sufficient material for 20 x 20µL reactions. Note: This Kit does not contain any NEDD8 E3 ligases.

Short Term Storage:	-80°C

Long Term Storage:	-80°C

Handling:	Avoid freeze/thaw cycles.

Miscellaneous/General:	This kit provides the means of generating a thioester-linked NEDD8-conjugated E2 enzyme, utilizing the first two steps in the NEDD8 cascade, for use in the NEDDylation of E3 ligases and target substrate proteins (user supplied). A NEDD8-specific antibody is provided for detection of NEDDylated proteins via SDS-PAGE and Western blotting. This kit provides sufficient material for 20 x 20µL reactions. Note: This Kit does not contain any NEDD8 E3 ligases. NEDD8 (neural precursor cell expressed developmentally downregulated gene 8) is an ubiquitin-like protein, with approximately 60% identity to ubiquitin (the highest homology of any ubiquitin-like protein). Conjugation of mature NEDD8 (via its exposed C-terminal glycine⁷⁶ residue) to specific lysine residues on a limited number of cellular target proteins via isopeptide bonds, allows NEDD8 to play a critical regulatory role in cell proliferation and development. The NEDD8 conjugation pathway proceeds by a mechanism analogous to that of the ubiquitin conjugation cascade, consisting of a dedicated NEDD8 E1 activating enzyme (a heterodimeric complex consisting of a regulatory subunit APP-BP1 and a catalytic subunit Uba3), E2 conjugating enzyme (UbcH12) and substrate specific E3 ligases. An increasing number of substrates for NEDDylation, including the tumor suppressors p53 and VHL, and EGFR have been identified in addition to members of the well-characterized cullin family of proteins that play a structural role in ubiquitin E3 ligase complexes such as SCF. Cullin NEDDylation in SCF complexes is facilitated by the RING domain containing Rbx1 (Roc1) SCF subunit, which is thought to act as a NEDD8 E3 ligase, and enhances the complex’s ubiquitin E3 ligase activity. NEDDylation of p53 is mediated by its ubiquitin E3 ligase Mdm2, providing an additional control mechanism for inhibition of its transcriptional activity. E3 independent NEDDylation of p53 has also been demonstrated in vitro. Reconstitution of the NEDD8 conjugation pathway in vitro using recombinant proteins is readily achieved and has been used to investigate a number of NEDD8 modification systems.

General Literature References

In vitro systems for NEDD8 conjugation by Ubc12: T. Chiba; Methods Enzymol. 398, 68 (2005), Abstract;

A hitchhiker’s guide to the cullin ubiquitin ligases: SCF and its kin: A.R. Willems, et al.; Biochim. Biophys. Acta 1695, 133 (2004), Abstract;

Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity: D.P. Xirodimas, et al.; Cell 118, 83 (2004), Abstract;

Nedd8 on cullin: building an expressway to protein destruction: Z.Q. Pan, et al.; Oncogene 23, 1985 (2004), Abstract;

Neddylating the guardian; Mdm2 catalyzed conjugation of Nedd8 to p53: J.W. Harper; Cell 118, 2 (2004), Abstract;

pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development: N.H. Stickle, et al.; Mol. Cell. Biol. 24, 3251 (2004), Abstract;

Ubiquitin-like protein activation: D.T. Huang, et al.; Oncogene 23, 1958 (2004), Abstract;

Nedd8-modification of Cul1 is promoted by Roc1 as a Nedd8-E3 ligase and regulates its stability: M. Morimoto, et al.; Biochem. Biophys. Res. Commun. 301, 392 (2003), Abstract;

Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha: M.A. Read, et al.; Mol. Cell. Biol. 20, 2326 (2000), Abstract;

Identification of the activating and conjugating enzymes of the NEDD8 conjugation pathway: L. Gong & E.T. Yeh; J. Biol. Chem. 274, 12036 (1999), Abstract;