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UbcH12 (human), (recombinant) (His-tag)

 
BML-UW9145-0100 100 µg 258.00 USD
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Product Specification

MW:~22kDa
 
Source:Produced in E. coli BL21 (λDE3). UbcH12 is fused to an N-terminal His-tag.
 
UniProt ID:P61081
 
Formulation:Liquid. In HEPES, pH 7.5, containing 100mM NaCl, 1mM DTT, 10% glycerol.
 
Purity:≥95% (SDS-PAGE)
 
Application Notes:Uses:
NEDD8 modification of specific proteins in vitro.
Demonstrate novel proteins are potential targets for NEDDylation under in vitro conditions.
Generate substrates for deNEDDylating enzymes.
NEDDylation pathway studies
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
Handling:Avoid freeze/thaw cycles. After opening, prepare aliquots and store at -80°C.
 
Scientific Background:NEDD8 (neural precursor cell expressed developmentally downregulated gene 8) is an ubiquitin-like protein, with approx. 60% identity to ubiquitin. 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, a process closely associated to the ubiquitin-proteasome pathway.

The NEDD8 conjugation pathway proceeds by a mechanism analogous to that of the ubiquitin conjugation cascade, consisting of a dedicated NEDD8 E1 activating enzyme (APP-BP1/Uba3), E2 conjugating enzyme (UbcH12) and substrate specific E3 ligases. An increasing number of substrates for NEDDylation, including the tumor suppressors p53 and VHL, have been identified in addition to members of the well characterised 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 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.  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.

UbcH12, the major NEDD8 E2 enzyme, conjugates activated NEDD8 and mediates its subsequent linkage, via C-terminal isopeptide bond formation, to a number of proteins, including the tumour suppressors p53 and VHL, in addition to its well characterised modification of members of the cullin ubiquitin E3 ligase family. Such modifications allow NEDD8 to play a critical regulatory role in cell proliferation and development, a process closely associated to the ubiquitin-proteasome pathway.
 
BML-UW9145 SDS-PAGE
SDS-PAGE analysis: Lane 1: MW marker, Lane 2: 2µg UbcH12 (human), (recombinant) (His-tag) (Prod. No. BML-UW9145).
BML-UW9145 WB
Western Blot Analysis: Lane 1: MW marker, Lane 2: 100 ng of UbcH12 (human), (recombinant) (His-tag) probed with Anti-6X His tag® antibody.
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BML-UW9145 SDS-PAGE BML-UW9145 WB

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. Kirodimas et al.; Cell 118, 83 (2004),
Nedd8 on cullin: building an expressway to protein destruction: Z.Q. Pan et al.; Oncogene. 23, 1985 (2004),
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),
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. : Gong, L. and Yeh, E. T.; J.Biol.Chem. 274, 12036-12042 (1999),

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