Ubiquitin Conjugation Rxn Buffer Kit
- 貨號 Cat. No.
- SK-10
- 規格 Size
- 1 kit
- 品牌 Brand
- Boston Biochem
Description
Ubiquitin Conjugation Rxn Buffer Kit
Product Information
1 kit
Use & Storage
Store at -20°C. Avoid multiple freeze/thaw cycles.
Catalog #: SK-10
Ubiquitin Conjugation Rxn Buffer Kit
Product Source:
Synthetic
Ubiquitin reaction buffer kit contains the optimal buffer formulations for use in conjugation assays of ubiquitin (U-100) to protein substrates in vitro. This requires the activities of the E1 activating enzyme (E-305) and subsequent E2 and E3 enzymes. This buffer can be used to supplement ubiquitin thioester/conjugation initiation kit (K-995).
Product Information
Quantity:
1 kit
Stock:
1. 1ml 10X Ubiquitin Reaction Buffer
2. 100μl 10X Mg-ATP
3. 1ml 100X E1 Stop Buffer
4. 1ml 5X Non-reducing gel loading buffer
Background:
The covalent bond between ubiquitin and substrate proteins requires the activation of the C-terminal carboxyl group of Ub to be activated. In this reaction, ubiquitin is linked to the Ubiquitin Activating Enzyme (E1) through a thioester bond between its C-terminal glycine and the active site cysteine residue in the enzyme (E1-S-Ub). E1 hydrolyzes ATP to AMP and PPi with the intermediate formation of an E1-bound ubiquitin adenylate. The activated Ub is then transferred from E1 to any of
a family of Ubiquitin Carrier Protein Enzymes (E2s) in a similar thioester linkage (E2-S-Ub). Ubiquitin can then be transferred directly to the ε-Lys of a substrate from E2 enzymes. Other conjugation reactions require the involvement of a third enzyme, Ubiquitin Ligase (E3). E3 binds to substrate proteins and also to an E2 to form an E2-E3-substrate complex. It is the recognition and formation of said complex that has the highest level of substrate specificity for the conjugation
cascade.
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.