UFM1 Conjugation Rxn Buffer Kit
- 貨號 Cat. No.
- SK-30
- 規格 Size
- 1 kit
- 品牌 Brand
- Boston Biochem
Description
UFM1 Conjugation Rxn Buffer Kit
Product Information
1 kit
Use & Storage
Store at -20°C. Avoid multiple freeze/thaw cycles.
Catalog #: SK-30
UFM1 Conjugation Rxn Buffer Kit
Product Source:
Synthetic
Ufm1 reaction buffer kit contains the optimal buffer formulations for use in assays for the conjugation of the ubiquitin-like modifier Ufm1 (UL-500) to protein substrates in vitro, which requires the activities of the human Ufm1 E1 activating enzyme (E-319) and the Ufc1 E2 enzyme (E2-675). This buffer can be used as a supplement to Ufm1 Conjugation Kit (K-500).
Product Information
Quantity:
1 kit
Stock:
1. 1ml 10X Ufm1 Reaction Buffer
2. 1ml 100X E1 Stop Buffer
3. 100μl 10X Mg-ATP
4. 1ml 5X Non-reducing gel loading buffer
Background:
The Ufm1 E1 enzyme uses ATP to adenylate the C-terminal glycine residue of Ufm1, forming a high-energy thiolester bond. The second step is the trans-esterification reaction whereby the activated Ufm1 is transferred to the active site cysteine of Ufc1. Ufc1 is a member of the E2 family and is homologous to ubiquitin-conjugating enzymes, but is specific for the conjugation of Ufm1 to a variety of target proteins. The Ufm1 pathway is distinct from ubiquitination with different substrate specificity and interaction with ligating enzymes. It is known that Ufm1 becomes conjugated to as yet unidentified proteins in HEK cells and various mouse tissues. Ufm1 is conserved in metazoa and plants, but not yeast. The exact cellular function and role of Ufm1 modification in vivo is not yet known.
Recommended Assay Protocol:
Protocol (20 μL reaction):
1. Add 2 μL reaction buffer (1X final) to appropriate enzyme and substrate mix.
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 minutes prior to loading on gel.
Use & Storage
Storage:
Store at -20°C. Avoid multiple freeze/thaw cycles.