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 specialty 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 |