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HTRF Human XIAP / BIR3 Binding Red Ligand

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HTRF Human XIAP / BIR3 Binding Red Ligand
Shipping box for Revvity reagent kits
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Shipping box for Revvity reagent kits
Shipping box for Revvity reagent kits
Shipping box for Revvity reagent kits
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HTRF XIAP BIR3 Red Ligand can be used for affinity binding experiments in association with the HTRF XIAP BIR3 Binding kit to investigate cooperativity effects within PROTAC drug discovery.

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

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March lab savings - SAVE 10%. Use promo code below.
MARCH10
Terms and conditions apply.
View product information
Product Variant
Unit Size: 1 unit
Part #:
64XIAPFLRED
List Price
USD 427.39
Your online price:
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, and disposal requirements under European REACH regulations (EC 1907/2006).
Total list price:
USD 427.39
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Shipping box for Revvity reagent kits
Shipping box for Revvity reagent kits
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HTRF Human XIAP / BIR3 Binding Red Ligand
Shipping box for Revvity reagent kits
Video Play Button
Shipping box for Revvity reagent kits
Shipping box for Revvity reagent kits
HTRF Human XIAP / BIR3 Binding Red Ligand
Shipping box for Revvity reagent kits
Shipping box for Revvity reagent kits
Video Play Button

Product information

Overview

The HTRF XIAP BIR3 Red Ligand is based on labeled A410099.1 derivative and is primarily intended to perform cooperativity binding studies. It enables calculation of the affinity of the XIAP BIR3 Red Ligand for XIAP BIR3 protein in the presence of the targeted protein of interest. The Kd of HTRF XIAP BIR3 Red Ligand for XIAP protein, in absence and presence of the targeted protein, enables an accurate analysis of the cooperative effect assessed with the HTRF XIAP BIR3 binding kit.

Specifications

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

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HTRF Human XIAP / BIR3 Binding Red Ligand
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HTRF Human XIAP / BIR3 Binding Red Ligand
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How it works

HTRF XIAP ligand binding assay principle on the XIAP BIR3 binding domain

HTRF XIAP BIR3 Red Ligand binding is detected in a direct binding assay format, using an anti GST-Terbium Cryptate antibody which binds to GST-tagged Human XIAP 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 XIAP BIR3 Red Ligand (Image B).

ppi-how-it-works-assay-principle-human-xiap-bir3-red-ligand
ppi-how-it-works-assay-principle-human-xiap-bir3-red-ligand
HTRF XIAP BIR3 Red Ligand binding assay protocol

Saturation binding experiments of HTRF XIAP BIR3-Red Ligand can be run in 384-well plates by dispensing 5 µL of Diluent 9 buffer (for Total binding) or XIAP 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 XIAP BIR3 protein. Finally, 5 µL of a pre-mixed solution of HTRF XIAP BIR3-Red Ligand and Anti-GST Tb cryptate antibody are added.

The HTRF Ratio is measured after 2 hours of incubation at room temperature

ppi-how-it-works-assay-protocol-human-xiap-bir3-red-ligand

 

Assay details

HTRF XIAP BIR3-Red Ligand Kd (reference without PROTAC protein substrate) 5 nM +/- 8nM

 

Assay validation

Determination of the affinity binding (Kd) of XIAP BIR3-Red Ligand in the presence of targeted protein

In this example, the affinity binding (Kd) of the XIAP BIR3 Red Ligand to GST-XIAP 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 XIAP 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 (XIAPBIR3 -PROTAC) by the Ki of the PROTAC in the ternary complex (XIAPBIR3 -PROTAC-BRD4).

assay-validation-human-xiap-bir3-red-ligand

 

Resources

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