DCAF1 Binding Assay Kit
The DCAF1 Binding Assay Kit is a fluorescence polarization (FP), homogeneous, 96-well assay kit designed for the screening and profiling of molecules that inhibit the binding of the DCAF1 (DDB1 and CUL4 Associated Factor 1) Complex to a high affinity DCAF1 fluorescent probe. The kit contains enough purified recombinant DCAF1/Rbx1 (M5I)/DDB1/CUL4A Complex, fluorescent probe and assay buffer for 100 reactions.
Figure 1: Illustration of the DCAF1 Binding Assay Kit principle.
The assay uses a DCAF1 fluorescent probe. This is a very small molecule that rotates fast (low FP). This probe is incubated with the DCAF1 complex leading to the formation of a large complex, with restricted movement (high FP). FP is proportional to binding of DCAF1 to the fluorescent probe.
This assay requires a fluorescent microplate reader capable of measuring fluorescence polarization (FP) and equipped with the required parts to read the FP signal. For more information FP technology, visit our Tech Note: FP, assay principles and applications.
Need us to run inhibitor screens or profile your compounds against DCAF1? Check out our Ubiquitination Screening and Profiling.
- Adjustable micropipettor and sterile tips
- Rotating or rocker platform
- Fluorescent microplate reader capable of measuring fluorescence polarization (λex=485/20 nm and detection at λem=528/20 nm)
| Catalog # | Name | Amount | Storage |
| 102190 | DCAF1/Rbx1 (M5I)/DDB1/ CUL4A Complex* | 100 µg | -80°C |
| 10 µM BDY DCAF1 Substrate | 15 µl | -80°C | |
| DCAF1 Assay Buffer (FP) | 10 ml | -20°C | |
| 79685 | Low binding, black 96-well plate | 1 | Room Temp |
* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.
DCAF1 (DDB1 and CUL4 Associated Factor 1), also known as VprBP (Vpr binding protein), is a WD40 repeat domain-containing E3 ligase of the CRL4 (cullin RING ligase 4) subfamily of proteins. It functions as the substrate recognition unit in E3 ligase complexes, such as the EED (embryonic ectoderm development)-DDB1 (DNA damage-binding protein)-VprBP (or EDVP) and the CRL4DCAF1 complex. The CRL4DCAF1 complex is composed of CUL4A (cullin-4A), DDB1 and DCAF1. DCAF1 is involved in various normal physiological functions in the cell, including cell cycle regulation/progression, cell division, lipid metabolism, and miRNA biogenesis. Additionally, DCAF1 plays an E3 ligase-independent role, by acting as a kinase and phosphorylating Histone H2A. Antagonists of DCAF1 could be used toward the development of therapeutics for cancer and viral treatments. DCAF1 can also be used to create bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of proteins of interest. For instance, the use of a DCAF1-BTK (Bruton’s tyrosine kinase) PROTAC® resulted in degradation of BTK in cells that had acquired resistance to CRBN (cereblon)-BTK PROTACs, making DCAF1 an interesting alternative E3 ligase in cases of resistance mechanism to other commonly used PROTACs. Design and characterization of DCAF1 binders is an important step in the design of DCAF1-targeting molecular degraders.
Li A., et al., 2023 J Med Chem 66 (7): 5041-5060.
Schröder M., et al., 2024 Nat Commun 15 (1): 275.
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