Description
TRF MI-1061 Red ligand is primarily intended to perform affinity binding curves to MDM2 protein using HTRF® technology.
MDM2, also known as murine double minute 2, is involved in many biological processes and is closely associated with DNA repair, cell cycle arrest, apoptosis, and the occurrence of many diseases. MDM2 is one of the most popular E3 ligases which is recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a targeted protein. MDM2 interacts with several proteins to form the functional E3 ubiquitin ligase complex,in which MDM2 functions as a substrate receptor of the E3 ubiquitin ligase complex and targets various proteins as targeted-protein for proteolysis.
Identifying new MDM2 ligands is therefore a way to control biological processes involved in oncology, neurodegenerative, or metabolic diseases by inducing the proteasomal-dependent degradation of proteins like p53.
HTRF MI-1061 Red ligand binding assays can be used in association with the HTRF MDM2 binding kit (#64BDMDM2PEG/H) for cooperativity binding studies to monitor the effect of PROTAC protein substrate on the Kd of HTRF MI-1061 Red ligand for MDM2 protein, enabling accurate analysis of cooperative effect monitored with the HTRF MDM2 binding kit.
HTRF MI-1061 ligand binding assay principle on MDM2 protein
HTRF MI-1061 Red Ligand binding is detected in a direct binding assay format using an anti GST-Europium Cryptate antibody which binds to GST-tagged Human MDM2. 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 Kd determination for HTRF MI-1061 Red Ligand (Image B).
HTRF MI-1061 Red Ligand binding assay protocol
Saturation binding experiments of HTRF MI-1061 Red Ligand can be run in 384-well plates by dispensing 5µL of Diluent 9 buffer (for Total binding) or MDM2 standard (for non specific binding). Then 5µL of PROTAC binding buffer 3(for reference) or PROTAC protein substrate diluted in PROTAC buffer binding buffer 3 (for cooperativity studies) are added, followed by 5µL of MDM2 GST-tagged protein. Finally, 5µL of a pre-mixed solution of HTRF MI-1061 Red Ligand and Anti-GST Eu cryptate antibody are added.
The HTRF ratio is measured after 1 hour of incubation at room temperature.
Example of cooperativity binding studies with HTRF MI-1061-Red Ligand
Example of cooperativity binding studies with HTRF MI-1061 Red Ligand to monitor the effect of a PROTAC protein substrate as model tested at 20nM, on the Kd of HTRF MI-1061 Red ligand with MDM2 GST-tagged protein in the same conditions as the PROTAC compound screening assays performed with the HTRF MDM2 binding kit. Results from this example show no effect of this PROTAC protein substrate on the Kd of HTRF MI-1061 Red ligand.
