Description
Key facts
Sample types:Purified protein,
Assay type:Enzyme activity
Product details:
E2-Ubiquitin Conjugation Kit (ab139472) provides the tools necessary for generating a range of thioester-linked ubiquitin-conjugated E2 enzymes for use in ubiquitinylation experiments. Biotinylated ubiquitin (provided in the kit) can be detected with streptavidin-linked enzymes via SDS PAGE and western blotting.
This product provides sufficient material for a total of 50 reactions: 4 reactions with each included E2 enzyme.
Uses for this kit include:
1. Ubiquitinylation of target proteins in presence of dedicated E3 ligase. Panel of E2s provided for generation of E2-Ub thioester conjugates for testing vs. specific E3/target combinations. For example: ubiquitinylation of p53 in the presence of mdm2 (E3) and UbcH5b (E2).
2. Activation of Ub for thioester conjugation to novel E2 enzymes (substituted like for like with kit E2s, under directly comparable conditions).
Ubiquitinylation, the covalent attachment of ubiquitin to proteins, is achieved through three enzymatic steps. In an ATP-dependent process, the ubiquitin activating enzyme (E1) catalyzes the formation of a reactive thioester bond with ubiquitin, in the presence of a Mg2+ cofactor, followed by its subsequent transfer to the active site cysteine of a ubiquitin carrier protein (E2). The specificity of ubiquitin ligation arises from the subsequent association of the E2-ubiquitin thioester with a substrate specific ubiquitin-protein isopeptide ligase (E3), which facilitates the formation of the isopeptide linkage between ubiquitin and its target protein.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions--80°C, Appropriate long-term storage conditions--80°C, Storage information--80°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Ubiquitin E2 also known as E2 ubiquitin-conjugating enzyme is essential in the ubiquitin-proteasome system mediating protein ubiquitination. It acts as a carrier transferring ubiquitin from E1 the ubiquitin-activating enzyme to E3 ligase the ubiquitin ligase facilitating the ubiquitin-conjugation process. Ubiquitin E2 proteins typically have molecular masses ranging from 14 kDa to 20 kDa. They distribute widely in eukaryotic cells playing roles across various tissues.
Biological function summary
Ubiquitin E2 enzymes contribute to protein degradation by tagging proteins for degradation by the 26S proteasome. They form a complex with E1 and E3 to ensure specific proteins are accurately marked for turnover. This precise marking regulates many cellular processes such as cell cycle progression DNA repair and response to oxidative stress.
Pathways
Ubiquitin E2 enzymes integrate into significant cellular pathways like the cell cycle and DNA damage response. They collaborate with other proteins such as cyclins and ubiquitin E3 ligases to regulate cell division. During DNA repair ubiquitin E2 works with RAD proteins to identify and process damaged DNA maintaining genomic stability.
Ubiquitin E2 enzymes associate with cancer and neurodegenerative diseases such as Parkinson's disease. Their dysregulation leads to abnormal protein accumulation or degradation contributing to disease pathogenesis. They interact with proteins like Parkin an E3 ligase which is significant in Parkinson's disease and p53 a tumor suppressor protein highlighting their importance in these disorders.
