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HTRF Thalidomide-Red Ligand HTRF®

HTRF Thalidomide-Red ligand is a derivative of Thalidomide and can be used as a fluorescent tracer for HTRF Thalidomide-Red ligand binding assays, associated with the HTRF Cereblon Binding kit to investigate cooperativity effects within PROTAC drug discovery.

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  • Highly specific Highly specific
  • No-wash No-wash
  • Validated with reference Ab Validated with reference Ab

HTRF Thalidomide-Red ligand is a derivative of Thalidomide and can be used as a fluorescent tracer for HTRF Thalidomide-Red ligand binding assays, associated with the HTRF Cereblon Binding kit to investigate cooperativity effects within PROTAC drug discovery.

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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 (#PerkinElmer 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.  

Benefits

  • EQUILIBRIUM BINDING
  • PHARMACOLOGICAL STUDIES
  • COOPERATIVITY EFFECT

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).

Principle of HTRF Thalidomide-Red ligand binding assays on Cereblon protein
Principle of HTRF Thalidomide-Red ligand binding assays on Cereblon protein

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.

Assay protocol for HTRF Thalidomide-Red ligand binding assays on Cereblon protein

HTRF Thalidomide-Red ligand Kd (reference without PROTAC protein substrate)

3.2nM ±0.8 (SD)

Example of cooperativity binding studies with HTRF Thalidomide-Red ligand

,Example of cooperativity binding studies with HTRF Thalidomide-Red ligand to monitor the effect of a PROTAC protein substrate as model tested at 100nM, on the Kd of HTRF Thalidomide-Red ligand with GST-CRBN protein in the same conditions as the PROTAC compound screening assays performed with the HTRF CRBN binding kit. Results from this example show no effect of this PROTAC protein substrate on the Kd of HTRF Thalidomide-Red ligand. 

Kd determination of HTRF Thalidomide-Red ligand in cooperativity binding studies

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