Nedd8 Conjugation Kit

Nedd8 Conjugation Kit is designed for assaying the conjugation of Nedd8 to protein substrates in vitro. In a protein neddylation reaction, Nedd8 is first activated by the Nedd8 activating enzyme APPBP1/UBA3 (E1) in an ATP hydrolysis-dependent manner; activated Nedd8 is then transferred to the Nedd8 conjugating enzyme Ube2M (UbcH12) (E2) by formation of a thiolester bond with the catalytic cysteine residue of UbcH12I; Nedd8-charged Ube2M (UbcH12) can mediate protein neddylation in the presence of an appropriate Nedd8 ligase (E3).

Additional Information

Product Name: Nedd8 Conjugation Kit
Also Known As: N/A
Catalog No.: J3150
Size: 1 Kit
Molecular Weight: N/A
Species: Human
Source: Bacterial recombinant
Stock: 20 mM Tris, 150 mM NaCl, 2 mM βME, 10% Glycerol
Concentration: See tube label
Quality Assurance: 95% based on SDS-PAGE
Storage: Store at -80°C; avoid multiple freeze-thaw cycles
PDF Data Sheet: PDF datasheet, MSDS
NCBI RefSeq: N/A
Image(s): No
Shipping Method: Dry ice shipping
References: N/A

Details

* This kit is sufficient for setting up 25 x 20 µl reactions.

Description

Nedd8 Conjugation Kit is designed for assaying the conjugation of Nedd8 to protein substrates in vitro. In a protein neddylation reaction, Nedd8 is first activated by the Nedd8 activating enzyme APPBP1/UBA3 (E1) in an ATP hydrolysis-dependent manner; activated Nedd8 is then transferred to the Nedd8 conjugating enzyme Ube2M (UbcH12) (E2) by formation of a thiolester bond with the catalytic cysteine residue of UbcH12I; Nedd8-charged Ube2M (UbcH12) can mediate protein neddylation in the presence of an appropriate Nedd8 ligase (E3).

A typical 20 µl reaction contains 100 nM APPBP1/6xHis-UBAs, 2 µM 6xHis-Ube2M (UbcH12), 15 µM 6xHis-Nedd8, 4 mM ATP, 2 µM E3 (not supplied) and 2 µM protein substrate (no supplied) in the Neddylation Buffer.

Components

• 20X APPBP1/6xHis-UBA3 (2 µM)................25 µl
• 20X 6xHis-Ube2M (UbcH12) (40 µM))..........25 µl
• 10X 6xHis-Nedd8 (150 µM)..........................50 µl
• 10X ATP (40 mM)........................................60 µl
• 10X Neddylation Buffer...............................100 µl