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InformationUbiquitin Activating Enzyme (E1, E1/UBAE, ubiquitin-activating) ELISA Kit (Part hE1UBAE-ELISA)
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Ubiquitin Activating EnzymeE1/UACE ELISA Kit
E1/ubiquitin-activating enzyme ELISA Kit
Dynamic Assay Range: 50pg/ml - 30,000pg/ml depending on species.
Lead Time: Approximately 1.5 weeks
UBAE ELISA Kit Availability and Specifications
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Species |
Standard |
Assay Range |
| Human | 25.6ng/ml | 100pg/ml - 30ng/ml |
| Rat | 24ng/ml | 50pg/ml - 20ng/ml |
| Mouse | 19.2ng/ml | 50pg/ml - 15ng/ml |
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Highlights
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Intoduction
to E1/UBAE
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Ubiquitin-activating enzyme, (or E1), is involved in the first step of ubiquitination. At the start of the ubiquitination cascade, the E1 enzyme binds ATP-Mg2+ and ubiquitin and catalyses ubiquitin C-terminal acyl adenylation. In the next step a catalytic cysteine on the E1 enzyme attacks the ubiquitin-AMP complex through acyl substitution, simultaneously creating a thioester bond and an AMP leaving group. Finally, the E1~ubiquitin complex transfers ubiquitin to an E2 enzyme through a transthioesterification reaction, in which an E2 catalytic cysteine attacks the backside of the E1~ubiquitin complex. Throughout this mechanism, the E1 enzyme is bound to two ubiquitin molecules. Although this secondary ubiquitin is similarly adenylated, it does not form the same thioester complex described previously. The function of the secondary ubiquitin remains largely unknown, however it is believed that it may facilitate conformational changes seen in the E1 enzyme during the transthioesterification process.
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Methods
Overview
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The included ELISA micro-strip plate is provided pre-coated with an a monoclonal capture antibody selective to the antigen. Samples are pre-mixed with a biotin-linked detection antibody and streptavidin-HRP, which are then added to the plate for 60 min. The plate is washed after a 60 min incubation period. Color reagents are added, resulting in a reaction with HRP that yields a blue colored solution. A stop solution is applied to terminate the reaction, and the solution is measured at 450nm.
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Sandwich ELISA Methods to E1/UBAE
ELISA Assay Steps
- Mix sample, biotinylated-detection antibody and streptavidin-HRP.
- Apply sample and antibody-HRP complex to ELISA plate for 60min at 37oC.Wash 5 times.
- Apply chromogens, incubate, then stop reaction.
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E1/UBAE
References 1. Haas, A. L., and I. A. Rose. 1982. "The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis." Journal of Biological
Chemistry 257.17,10329-10337.
2. Haas, Arthur L., et al. 1982. "Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation." Journal of Biological Chemistry 257.5, 2543-2548.
3. Handley, Patricia M., et al. 1991. "Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme E1." Proceedings of the National Academy of Sciences 88.1, 258-262.
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For research only. Not for diagnostics.
Safety: Stop solution contains acid. Avoid ingestion, skin and eye contact.
Storage: 4°C. 6 months.
Shipping. Overnight domestic delivery recommended with ice pack (cost: $52 for 1-2 kits). Also avaialble for international delivery; please inquire for shipping cost.
