Revvity, 64BDCRBNRED, HTRF Thalidomide Red Ligand

Overview
HTRF Thalidomide-Red ligand is primarily intended to perform affinity binding curves to Cereblon protein using HTRF® technology.
Cereblon, also known as CRBN, is involved in many biological processes and is closely associated with proliferation, apoptosis, cell metabolism, and the occurrence of many diseases. Cereblon is also one of the two most popular E3 ligases which is recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a targeted protein. Cereblon interacts with several proteins to form the functional E3 ubiquitin ligase complex, in which CRBN functions as a substrate receptor of the E3 ubiquitin ligase complex and targets proteins for proteolysis.
HTRF Thalidomide-Red ligand binding assays can be used in association with the HTRF Cereblon binding kit (#64BDCRBNPEG/H) for cooperativity binding studies in order to monitor the effect of PROTAC protein substrate on the Kd of HTRF Thalidomide-Red ligand for Cereblon protein, enabling accurate analysis of cooperative effect monitored with the HTRF Cereblon binding kit.
How it works
HTRF Thalidomide-Red ligand binding assay principle on cereblon protein
HTRF Thalidomide-Red ligand binding is detected in a direct binding assay format using an anti GST-Europium Cryptate antibody which binds to Human Cereblon WT GST-tagged. When the dyes are in close proximity, the excitation of the donor with a light source (laser or flash lamp) triggers a Fluorescence Resonance Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at a specific wavelength (665 nm) (Image A). The specific binding signal is calculated by subtracting the non-specific binding signal from the total signal, enabling determination of Kd for HTRF Thalidomide-Red ligand (Image B).

 

HTRF Thalidomide-Red ligand binding assay protocol
Saturation binding experiments of HTRF Thalidomide-Red ligand can be run in 384-well plates by dispensing 5µL of Diluent 9 buffer (for Total binding) or Cereblon binding standard (for non specific binding). Then 5µL of PROTAC binding buffer 1 (for reference) or PROTAC protein substrate diluted in PROTAC buffer 1 (for cooperativity studies) are added, followed by 5µL of GST-CRBN protein. Finally, 5µL of a pre-mixed solution of HTRF Thalidomide-Red ligand and Anti-GST Eu cryptate antibody are added. The HTRF ratio is measured after 3 hours of incubation at room temperature.
The HTRF Ratio is measured after 2 hours of incubation at room temperature. Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Product Group-Fluorescent Reagent
Shipping Conditions-Shipped in Dry Ice
Technology-TR-FRET
Therapeutic Area-Inflammation Neuroscience Oncology & Inflammation
Unit Size-1 unit