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InformationUBIQUITIN LIGASE (E3) ELISA Kit (Ubiquin ligating enzyme; Part hE3UBPL-ELISA)
 ELISA kit Fivephoton Biochemicals-600x600.jpg "UBIQUITIN LIGASE (E3) ELISA Kit (Ubiquin ligating enzyme; Part hE3UBPL-ELISA)") Click to enlarge |
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Ubiquitin Ligase (E3 Ligase, UBPL) ELISA Kit
Human E3/ubiquitin-protein Ligase ELISA Kit
Dynamic Assay Range: 100pg/ml - 40,000pg/ml depending on species. Sensitivity: 42 pg/ml.
Lead Time: Approximately 1.5 weeks
UBPL ELISA Kit Availability and Specifications
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Species |
Standard |
Assay Range |
| Human | 16ng/ml | 50pg/ml - 15ng/ml |
| Rat | 40ng/ml | 100pg/ml - 38ng/ml |
| Mouse | 48ng/ml | 100pg/ml - 40ng/ml |
Icon Image (PDB). Ubiquitin ligase (blue) in complex with ubiquitin activating enzyme (cyan) and substrate.
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Highlights
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Intoduction
to E3/UBPL
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Ubiquitin ligase is the last enzyme in the cascade involved in the conjuation of ubiqutin to lysine. It operates in a complex with E2 ubiqutin acitvating enzyme. which is located in the cytosol, and attaches ubiquitin to lysine thorugh an isopeptide bond. The E3 enzyme is involved in the attachment of polyubiqutiin that targets proteins to degradation in the proteasome. The ubiqitin conjugating machinery is cytoplasmic and can be extracted for ELISA using a non-denaturing cell lysis bufer such as Part ELSP-1, or similar that releases a soluble cell lysate. The protocol for Part ELSP-1 provides a method to isolate a fraction with E3 for ELISA analysis.
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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. 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 Quantify Ubiquitin Ligase
ELISA Assay Steps
- Mix sample, biotinylated-detection antibody and streptavidin-HRP.
- Apply sample and antibody-HRP complex to ELISA plate for 60min at 37oC. Afterwards, wash 5 times.
- Apply chromogens, incubate, then stop reaction.
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E3 References 1. Robinson, P. A., and H. C. Ardley. 2004. "Ubiquitin-protein ligases." Journal of cell science 117.22, 5191-5194.
2. Shimura, Hideki, et al. 2000. "Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase." Nature genetics 25.3, 302-305.
3. Yang, Yili, et al. 2000. "Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli." Science 288.5467, 874-877.
4. Hershko, AHHESaCA, et al. 1983. "Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown." Journal of Biological Chemistry 258.13, 8206-8214.
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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 packs. Also avaialble for international delivery; please inquire for shipping cost.
