ISG15 Conjugation Rxn Buffer Kit
- 貨號 Cat. No.
- SK-25
- 規格 Size
- 1 kit
- 品牌 Brand
- Boston Biochem
Description
ISG15 Conjugation Rxn Buffer Kit
Product Information
1 kit
Use & Storage
Store at -20°C. Avoid multiple freeze/thaw cycles.
Catalog #: SK-25
ISG15 Conjugation Rxn Buffer Kit
Product Source:
Synthetic
ISG15 reaction buffer kit contains the optimal buffer formulations for use in assays for the conjugation of the ubiquitin-like modifier ISG15 (UL-601) to protein substrates in vitro, which requires the activities of the human ISG15 E1 activating enzyme (E-308, E-309) and the UbcH8 E2 enzyme (E2-644). This buffer can be used as a supplement to ISG15 Conjugation Kit (K-600).
Product Information
Quantity:
1 kit
Stock:
1. 1ml 10X ISG15 Reaction Buffer
2. 1ml 100X E1 Stop Buffer
3. 100μl 10X Mg-ATP
4. 1ml 5X Non-reducing gel loading buffer
Background:
The ubiquitin-like ISG15 is conjugated to a variety of proteins in the presence of UbcH8 and an E1 activating enzyme. The ISG15 E1 enzyme uses ATP to adenylate the C-terminal glycine residue of ISG15, forming a high-energy thiolester bond. The second step is the trans-esterification reaction whereby the activated ISG15 is transferred to the active site cysteine of UbcH8. UbcH8 is a member of the E2 family and is homologous to ubiquitin-conjugating enzymes, but is specific for the conjugation of ISG15 to a variety of target proteins. The ISG15 pathway is distinct from ubiquitination with different substrate specificity and interaction with ligating enzymes. ISG15 becomes conjugated to a diverse set of proteins after IFN-α/β stimulation or microbial challenge. The functions or biochemical consequences ISG15 conjugation to proteins are not yet known, but it appears that this modification does not target proteins for proteasomal degradation. ISG15 shows specific chemotactic activity towards neutrophils and activates them to induce release of eosinophil chemotactic factors. It may also serve as a trans-acting binding factor directing the association of ligated target proteins to intermediate filaments; and may also be involved in autocrine, paracrine and endocrine mechanisms.
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.