CREBBP TR-FRET Assay Kit
The CREBBP TR-FRET Assay Kit is designed to measure binding activity of CREBBP (cAMP response element-binding protein binding-binding protein) to its substrate for screening and profiling applications using TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer). It utilizes Terbium-labeled donor and Cy5-labeled acceptor to complete the TR-FRET pairing. The CREBBP TR-FRET Assay Kit comes in a convenient 384-well format, with enough purified CREBBP (amino acids 1081-1197), Ligand, Tb-Labeled Donor and Dye-Labeled Acceptor, BET Bromodomain Ligand and assay buffer for 384 reactions. The assay also includes Non-Acetylated Ligand 1 as control for the specificity of binding.
Figure 1: Illustration of the assay principle.
A sample containing terbium-labeled donor, dye-labeled acceptor, CREBBP, substrate, and an inhibitor is incubated for 2 hours. The fluorescence intensity is then measured using a fluorescence reader. In the presence of binding of CREBBP to its ligands, energy transfer occurs due to the proximity of the donor and acceptor. Disruption of the binding results in decrease of energy transfer. Fluorescence intensity at λ=665 nm corresponds directly to the binding of CREBBP to BET Bromodomain Ligand.
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- Fluorescent reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
- Adjustable micropipettor and tips
| Catalog # | Component | Amount | Storage |
| 31128 | CREBBP (KAT3A), GST-Tag* | 10 µg | -80°C |
| 33000 | BET Bromodomain Ligand | 2.72 µg | -80°C |
| 33005 | Non-Acetylated Ligand 1 | 0.78 µg | -80°C |
| Tb-Labeled Donor | 2 x 10 µl | -20°C | |
| Dye-Labeled Acceptor | 2 x 10 µl | -20°C | |
| 33012 | 3x BRD TR-FRET Assay Buffer 1 | 4 ml | -20°C |
| 79969 | White, Nonbinding, low volume, microtiter plate | 1 | Room Temp. |
* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.
CREBBP, also known as CREB-binding protein and cAMP response element-binding protein-binding protein, CBP or KAT3A, is a transcriptional coactivator. It has acetyltransferase activity, adding acetyl groups to histones and transcription factors, and serves as a protein scaffold in complex formation, thereby regulating gene transcription. It is ubiquitously expressed and interacts with multiple transcription factors. CREBBP has been implicated in several cancer types, including colorectal cancer and squamous cell carcinoma, but also diabetes, and neurological diseases such as Alzheimer’s disease (AD). The broad spectrum of roles it performs by interacting with specific transcription factors has made CREBBP an attractive clinical target. CREBBP has also been identified as a radiosensitizer, with inhibition of this protein resulting in higher sensitivity to radiation in CREBBP mutant tumors and cell lines potentially due to homologous recombination impairment. A deeper understanding of the role of CREBBP in several pathways and diseases, combined with the development of inhibitors for each of the CREBBP/partner interactions, will result in significant advances in cancer therapy.
Filippakopoulos P., et al., 2012 Cell 149: 214.
Kumar M., et al., 2021 Nature Communications 12: 6340.
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