Fraction II HeLa Conjugation Kit
- 貨號 Cat. No.
- K-930
- 規格 Size
- 1 kit
- 品牌 Brand
- Boston Biochem
Description
Fraction II HeLa Conjugation Kit
HeLa
Product Information
1 kit
Use & Storage
Enzyme/Fraction/Protein Solutions should be stored at -80°C. Avoid multiple freeze/thaw cycles.
Catalog #: K-930
Fraction II HeLa Conjugation Kit
Product Source:
HeLa
This kit contains reagents to allow for the controlled conjugation of exogenously added or endogenously contained substrate proteins through the ubiquitin proteasome pathway (UPP). Fraction II HeLa is the protein fraction of a cell extract that binds to anion exchange resin. It is essentially free of ubiquitin and ATP. FII contains E1, most E2s, some E3s, UCHs and the proteasome (20S and 26S).
NOTE: Kit contains reagents sufficient for 10 x 50 μl reactions.
Product Information
Quantity:
1 kit
Stock:
1. Fraction II HeLa, 250 μL
[HeLa] = X mg/ml in 50 mM HEPES, pH 7.6, 1 mM DTT
2. Ubiquitin, 10 X stock, 75 μL
[Ub] = X mg/ml in 50 mM HEPES, pH 7.6
3. Energy Regeneration Solution (ERS), 10X, 50 μL
4. Ubiquitin Aldehyde, 20 μg, solid
5. MG-132, Inhibitor Control, 20 μL
[MG-132] =X μM in 100% DMSO
Background:
The Ubiquitin Proteasome Pathway (UPP) is the cell’s principle mechanism for protein catabolism. The UPP has been shown to have significant involvement in the regulation of critical cellular processes such as transcription, oncogenesis, cell cycle progression, development, growth, selective elimination of abnormal proteins, and antigen processing.
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 Gly 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:
Assay considerations:
Fraction II HeLa is the protein fraction of a cell extract that binds to anion exchange resin. It is essentially ubiquitin- and ATP-free; FII contains E1, most E2s, UCHs, and the proteasome (20S and 26S). Conjugation of the protein of interest, if already contained in the HeLa fraction may be tracked via immuno-detection or if added exogenously, by immuno-detection or radio-label. When tracking the increase of ubiquitinated products in a SDS-PAGE band is your approach, care must be taken to add substrate to an amount that gives a clearly defined signal. The ultimate optimal amount of substrate to be added must be determined experimentally. If the substrate protein is already contained in the Fraction II HeLa and is being assayed by immunodetection, this is not an issue.
The following assay protocol is for the set-up and execution of the experimental protein conjugation. Final concentrations of substrate and time courses must be determined experimentally for each individual protein. Detection methods must also be optimized based on activity, substrate quantity and time. The removal of ERS, ubiquitin or inhibitors are viable
negative controls for demonstration of a UPP mediated event.
Use & Storage
Storage:
Enzyme/Fraction/Protein Solutions should be stored at -80°C. Avoid multiple freeze/thaw cycles.