HTRF LCL161 - Red Ligand

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HTRF LCL161 - Red Ligand

HTRF LCL161-Red Ligand can be used for affinity binding experiments in association with the HTRF cIAP1 BIR3 Binding kit to investigate cooperativity effects within PROTAC drug discovery.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).

Product information

SDS, COAs, Manuals and more

Product information

Overview

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.

Specifications

Other Specifications

Application	Protein Detection
Assay Points	1
Assay Target Type	Fluorescent reagent
Assay Technology	HTRF
Automation Compatible	Yes
Brand	HTRF
Detection Method	HTRF
Experimental Type	In vitro
Quantity	1
Therapeutic Area	Inflammation Neuroscience Oncology & Inflammation
Unit Size	1 Unit

Video gallery

How it works

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).

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2ppi-how-it-works-assay-principle-htrf-lcl161-red-ligand-2.svg

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.

Assay details

HTRF LCL161 - Red Ligand Kd (reference without PROTAC protein substrate)

12 nM ± 10 (2SD)

Assay validation

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).