c-CBL TR-FRET Assay Kit
Casitas B lineage lymphoma (c-Cbl) is a multifunctional protein with ubiquitin E3 ligase activity capable of degrading diverse sets of proteins. c-Cbl is a ubiquitously expressed mammalian protein that plays a vital role in fundamental cellular functions including cell survival, migration, and proliferation. Recent evidence suggests a critical role for c-Cbl in angiogenesis and human solid organ tumors, making it a potential drug target in cancer immunotherapy. The c-CBL TR-FRET Assay Kit is designed to measure c-CBL auto-ubiquitination activity in a homogeneous 384 reaction format. It utilizes Biotin-labeled Ubiquitin and a terbium-labeled anti-c-CBL antibody to complete the TR-FRET pairing. This FRET-based assay requires no time-consuming washing steps, making it especially suitable for high throughput screening applications.
Figure: Illustration of Assay Principle
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Fluorescent microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
Adjustable micropipettor and sterile tips
| Catalog # | Component | Amount | Storage | |
| 80301 | UBE1 (E1)* | 25 µg | -80°C | Avoid freeze/ thaw cycles! |
| 80314 | UBCH5b (E2)* | 200 µg | -80°C | |
| 100370 | Human c-CBL (E3), GST-tag* | 10 µg | -80°C | |
| Biotin-Ubiquitin | 400 µl | -80°C | ||
| ATP (4 mM) | 1 ml | -80°C | ||
| U2 Assay Buffer** | 2 x 10 ml | -80°C | ||
| Tb-labeled donor | 10 µl | -20°C | ||
| Dye-labeled acceptor | 10 µl | -20°C | ||
| 79969 | White 384-well microtiter plate | 1 | Room Temp. |
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| Plate sealer | 1 | Room Temp. |
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*The concentrations of E1, E2, and E3 enzymes are lot-specific and will be indicated on the tubes containing the enzyme.
**In February 2023, buffer was re-named, but formulation unchanged
1. Lyle, C.L., et al., Cell. 2019; 8(5): E498.
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