(1) Standard curve
As can be seen from the graph, due to the competition between Thalidomide standard and Thalidomide-Ac for binding to CRBN/DDB1, the signal value decreases.
Product Details
Product Details
Product Specification
| Host | Human |
| Stability & Storage | -80°C |
Background
The Human Cereblon (CRBN) Protein Ligand Binding Assay Kit utilizes homogeneous time-resolved fluorescence (TR-FRET) technology and can be used for the binding detection and screening of CRBN inhibitors or ligands. It enables high-throughput screening for rapid detection of CRBN inhibitors or ligands, with the basic principle illustrated in the figure below. The Eu-labeled detection reagent (Anti-Tag1-Eu) specifically recognizes and binds to the Tag1-CRBN/DDB1 protein, while the receptor-labeled thalidomide (Thalidomide-Ac) binds to CRBN, bringing Eu and Ac into close proximity. Under excitation by an external light source, energy resonance transfer occurs between the donor and acceptor, and the signal intensity at 665 nm can be measured to determine the binding of CRBN/DDB1 to Thalidomide-Ac. The addition of CRBN ligands or PROTACs to the reaction system competitively binds to CRBN/DDB1, reducing the signal. As the concentration of ligands or PROTACs increases, the signal decreases. This homogeneous assay is simple to perform and does not require washing steps.

Components
Component |
Concentration |
Volume(100T) |
Volume(500T) |
Volume(2500T) |
Volume(10000T) |
Storage Condition |
|---|---|---|---|---|---|---|
Thalidomide |
10 mM in DMSO |
5 μL |
5 μL |
25 μL |
100 μL |
-80℃ |
Tag1-CRBN/DDB1 |
100× |
5μL |
25μL |
125μL |
500 μL |
-80℃ |
Thalidomide-Ac |
100× |
5μL |
25μL |
125μL |
500 μL |
-80℃ |
Anti-Tag1-Eu |
50× |
10μL |
50μL |
250 μL |
1000 μL |
-80℃ |
Detection Buffer |
10× |
250μL |
1.2mL |
6mL |
21 mL |
-80℃ |
*Reagents should be protected from repeated freeze-thaw cycles. It is recommended to aliquot after the first thaw and store according to the storage conditions. The shelf life is one year.
*Thaw reagents on ice before use and equilibrate to room temperature.
*Prepare all reagent working solutions freshly before use.
Picture
Picture
Bioactivity
(2) Validation of activity and inhibitors
Validation of the binding of Thalidomide-Ac to CRBN/DDB1. The results show that when the protein concentration remains constant, the detection signal increases as the concentration of Thalidomide-Ac rises. Lenalidomide, Pomalidomide, and PROTAC BET Degrader-1 can compete with Thalidomide-Ac for binding to CRBN/DDB1.
