Proteolysis Targeting Chimeras (PROTACs) have emerged as novel therapeutics to target the undruggable proteome. PROTACs are heterobifunctional small molecules that both simultaneously bind to a target protein and a ubiquitin (Ub) E3 ligase. This subsequently leads to ubiquitination and degradation of the target protein. The In vitro ubiquitination kit has been developed to establish a high throughput approach that can accurately predict PROTAC efficiency by monitoring the protein’s intrinsic ability to get ubiquitinated. We offer this kit for three E3 ubiquitin ligases: Cereblon, VHL, and HDM2 to monitor PROTAC mediated ubiquitination for target of choice.
New assay plate developed for colorimetric detection allows for quantitative measurement using absorbance-based plate readers. Colorimetric readouts (650nm, 480nm) are more consistent day-to-day than luminescent-based detection methods, fast, and easy to use.
For evaluating molecular glues, we recommend using our MG780: Molecular Glue In Vitro Ubiquitination Assay Kit. This kit is specially designed to measure the performance of molecular glues In vitro.
PROTAC In Vitro Ubiquitination Assay Kit Components
| In Vitro ubiquitination Assay Plate | 1 plate |
| Assay Buffer (10X) | 1.2mL |
| E3 Mix (20X) | 1 X 250µL |
| E1-E2-Ubiquitination Mix (20X) | 1 X 250µL |
| Secondary Antibody (Anti-Mouse or Anti-Rabbit HRP conjugate) | 60µL (100X) |
| Colorimetric Reagent (CR)Stopping Reagent (SR) | CR- 5mLSR- 5mL |
| Blocking Concentrate (5X) | 5mL |
| PROTAC in DMSO | 10µL |
| Target Protein positive control BRD3 (20X) | 20µL |
| Anti-BRD3 antibody (100X) | 5µL |
| Anti-mouse HRP Conjugate (100X) | 5µL |