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HTRF Human XIAP / BIR3 Binding Red Ligand
HTRF Human XIAP / BIR3 Binding Red Ligand
Revvity Vial from a Kit, Standard, or Control lysate
Product variant
Unit Size: 1 unit
Part #:
64XIAPFLRED
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).
HTRF Human XIAP / BIR3 Binding Red Ligand
Revvity Vial from a Kit, Standard, or Control lysate
HTRF Human XIAP / BIR3 Binding Red Ligand
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.
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).
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
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).
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
|
Video gallery
HTRF Human XIAP / BIR3 Binding Red Ligand
HTRF Human XIAP / BIR3 Binding Red Ligand
Resources
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Technical Note
How to access true Kd values in Protein-Protein Interaction assays
Investigate the dynamics of Interactions
Kinetic values are of importance for characterizing protein-protein interactions...
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