Description
HTRF LCL161-Red Ligand is primarily intended to perform cooperativity binding studies. It enables to calculate the affinity of the LCL161 Red Ligand for cIAP1 BIR3 protein in the presence of the targeted protein of interest. The obtained Kd of HTRF LCL161-Red Ligand for cIAP1 protein, in absence and presence of the targeted protein enables accurate analysis of cooperative effect assessed with the HTRF cIAP1 BIR3 binding kit.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
HTRF LCL161 ligand binding assay principle on the cIAP1 BIR3 binding domain
HTRF LCL161 - Red Ligand binding is detected in a direct binding assay format using an anti GST-Europium Cryptate antibody which binds to GST-tagged Human cIAP1 BIR3 domain. 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 the HTRF LCL161 - Red Ligand (Image B).
HTRF LCL161 - red ligand binding assay protocol
Saturation binding experiments of HTRF LCL161 - Red Ligand can be run in 384-well plates by dispensing 5 µL of Diluent 9 buffer (for Total binding) or cIAP1 BIR3 Binding standard (for non specific binding). Then 5 µL of PROTAC Binding Buffer 2 (for reference) or the targeted protein diluted in PROTAC Binding Buffer 2 (for cooperativity studies) are added, followed by 5 µL of GST-tagged cIAP BIR3 protein. Finally, 5 µL of a pre-mixed solution of HTRF LCL161 - Red Ligand and Anti-GST Tb cryptate antibody are added.
The HTRF Ratio is measured after 2 hours of incubation at room temperature.
Determination of the affinity binding (Kd) of LCL161-red ligand in the presence of targeted protein
In this example, the affinity binding (Kd) of the LCL161-Red Ligand to GST-cIAP1 BIR3 protein was assessed in the absence or presence of the targeted protein BRD4 (10nM or 100nM) . This result indicates that the presence of BRD4 does not change the Kd of the Red Ligand.
Consequently this example shows that a cooperativity experiment with a PROTAC molecule composed of a cIAP ligand and a BRD4 warhead can be set up. Finally, the alpha factor can be established by dividing the Ki of the PROTAC in the binary complex (cIAPBIR3 -PROTAC) by the Ki of the PROTAC in the ternary complex (cIAPBIR3 -PROTAC-BRD4).
