K-800

Material Data Sheet

NEDD8 Conjugation Initiation Kit

Price: $295.00
Catalog #: K-800

NEDD8 Conjugation Initiation Kit

Price: 
$295
Quantity: 
1 kit
Data Sheet: 
application/pdf iconK800_Nedd8 Conj Kit.pdf
Supplied: 
Protein
ConcentrationQuantity
10X NEDD8 E1 Enzyme
X mg/ml (X μM)20 μL
10X NEDD8
X mg/ml (X μM)20 μL
10X UbcH12
X mg/ml (X μM)20 μL
10X Mg-ATP SolutionX mM20 μL
 10X Reaction BufferX mM Hepes pH 820 μL

X mM NaCl
 

  
Background: 
The ubiquitin-like NEDD8 is conjugated to a variety of proteins in the presence of UbcH12 and an E1 activating enzyme. The NEDD8 E1 enzyme uses ATP to adenylate the C-terminal glycine residue of NEDD8, forming a high-energy thiolester bond. The second step is the trans-esterification reaction whereby the activated NEDD8 is transferred to the active site cysteine of UbcH12. UbcH12 is a member of the E2 family and is homologous to ubiquitin-conjugating enzymes, but is specific for the<br>conjugation of NEDD8 to a variety of target proteins. Nedd8 plays a critical regulatory role in cell proliferation and development, and modifies nearly all members of the Cullin family.

This kit is designed for the conjugation of the ubiquitin-like modifier NEDD8 to proteinsubstrates in vitro, which requires the activities of the heterodimeric human E1 activating enzyme(APPBP1/Uba3) and the UbcH12 E2 enzyme. The E1enzyme charges the NEDD8 by forming an ATP-dependent high energy thiolester bond with the active site cysteine of Uba3. The activated NEDD8 is subsequently transferred to UbcH12and this E2-S-Ub thiolester complex can be used for the conjugation of NEDD8 to protein substrates in the presence of the appropriate E3 enzymes (not supplied).

NOTE: Kit contains reagents sufficient for 10 x 20 μl reactions.

Storage: 
Store at -80°C. Avoid multiple freeze/thaw cycles.

Literature

References: 

Gong L., et al. (1999) J. Biol. Chem. 274: 12036-12042
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Kumar S., et al. (1993) Biophys. Biochem. Res. Comm. 195:393-399
Morimoto M., et al. (2003) Biophys. Biochem. Res. Comm. 301:392-398
Wada H., et al. (1999) Biophys. Biochem. Res. Comm. 275:100-105
Whitby F.G., et al. (1998) J. Biol. Chem. 273: 34983-34991