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NEDDylation Kit

 
BML-UW0590-0001 20 tests 366.00 USD
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  • 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

Application Notes: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.
 
Handling:Avoid freeze/thaw cycles.
 
Shipping:Shipped on Dry Ice
 
Short Term Storage:-80°C
 
Long Term Storage:-80°C
 
Kit/Set Contains:1. NEDD8 Activating Enzyme Solution (E1)
2. UbcH12 Solution (E2)
3. NEDD8 Solution
4. NEDDylation Buffer
5. Mg-ATP Solution
6. NEDD8 Antibody Solution
7. Non-reducing Gel Loading Buffer
 
Scientific Background: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.
 
UniProt ID:Q15843
 

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