NEDD8 Conjugation Rxn Buffer Kit
- 貨號 Cat. No.
- SK-20
- 規格 Size
- 1 kit
- 品牌 Brand
- Boston Biochem
Description
NEDD8 Conjugation Rxn Buffer Kit
Product Information
1 kit
Use & Storage
Store at -20°C. Avoid multiple freeze/thaw cycles.
Catalog #: SK-20
NEDD8 Conjugation Rxn Buffer Kit
Product Source:
Synthetic
NEDD8 reaction buffer kit contains the optimal buffer formulations for use in assays for the conjugation of the ubiquitin-like modifier NEDD8 (UL-812) to protein substrates in vitro. This requires the activities of the heterodimeric human E1 activating enzyme (APPBP1/Uba3) (E-313) and the UbcH12 E2 enzyme (E2-656). This buffer can be used to supplement NEDD8 conjugation kit (K-800).
Product Information
Quantity:
1 kit
Stock:
1. 1ml 10X NEDD8 Reaction Buffer
2. 100μl 10X Mg-ATP
3. 1ml 100X E1 Stop Buffer
4. 1ml 5X Non-reducing gel loading buffer
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 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.
Recommended Assay Protocol:
Protocol (20 μL reaction):
1. Add 2 μL reaction buffer (1X final) to appropriate enzyme and substrate.
2. Bring the volume to a total of 18 μL with dH2O if needed.
3. Initiate reaction with the addition of 2 μL of Mg-ATP solution (1 mM final).
4. Incubate the reaction at desired temperature and time.
5. Dilute 100X stop buffer in dH2O to 10X and stop reaction by adding 2 μL of 10X stop buffer.
3. Add 4 μL 5X loading buffer for SDS-PAGE/Western Blot. For reducing conditions, add 1mM β-mercaptoethanol or DTT (fresh) and boil for 5 min. prior to loading on gel.
Use & Storage
Storage:
Store at -20°C. Avoid multiple freeze/thaw cycles.