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Invitrogen™ Predictor™ hERG Fluorescence Polarization Assay Kit

Catalog No. PV5365
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Description

Predictor™ hERG Fluorescence Polarization Assay Kit

The Predictor™ hERG Fluorescence Polarization Assay Kit provides a set of validated components to perform hERG channel biochemical binding studies in the absence of radioligand. The assay was designed to identify potential hERG channel blockers by producing data that accurately correlates with patch-clamp electrophysiology studies. The assay is based on the principle of fluorescence polarization where a red-shifted fluorescent tracer is displaced from the hERG channel by compounds that bind to the channel. Assay performance was validated with a panel of established hERG channel blockers. Results from the Predictor™ assay demonstrate a high correlation with those obtained from patch clamp techniques.

Specifications

Color Red
Conjugate Predictor hERG Tracer Red
Detection Method Fluorescence
For Use With (Equipment) Microplate Reader
Quantity 1 Kit
Packaging 384-well Plate
Content And Storage

PV5365 Predictor™ hERG Fluorescence Polarization Assay Kit
(Other materials are required separately; please refer to the protocol)
Includes:
• Predictor™ hERG Membranes
• Predictor™ hERG Tracer Red
• Predictor™ hERG Buffers
• E-4031
• Predictor™ hERG Fluorescence Polarization Assay Protocol
• Certificate of Analysis
Store the Predictor™ hERG Membranes at -80°C. The Predictor™ Tracer Red, Predictor™buffers, and E-4301 should be stored at -20°C.
Assay Entry Biochemical binding
Druggable Target Ion Channels
Gene Symbol hERG
Readout End Point
Shipping Condition Dry Ice
System Type Predictor™ System
Target Entry hERG
Product Line Predictor
Protein Family Ion Channels
Product Type Assay Kit
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Frequently Asked Questions (FAQs)

I am doing drug discovery. Can I use the Predictor hERG Membranes for radioligand binding?

No, the Predictor hERG Membranes are not suitable for radioligand binding.
These membranes contain extremely high levels of hERG channel protein. Radioligand binding assays on these membranes would require either abnormally high concentrations of radioligand or severe dilutions of the membrane preparation, which tend to have a deleterious effect on hERG channel conformation and stability.
What are the common issues encountered with FP assays?

-No assay window: Check that your instrument is designed to do FP experiments.
-Poor discrimination between the minimum and maximum mP Control (both include the receptor): This may be an indication of the quality of the receptor. Do not make single use aliquots of the receptor or store diluted.
-The type of microplate is critical: Please see if the protocol specifies untreated polystyrene or NBS-coated plates. If the plate binds the fluorescent substrate, it cannot freely rotate and the mP reading will always be high. White plates cannot be used for FP assays. Avoid the use of silanized pipette tips and vials.
-The “No Receptor Control' (free Fluormone/Tracer) should have much higher RFU values compared to buffer alone. If not, there is a problem with the instrument setup or less commonly with the Fluormone/Tracer.
-If the free Fluormone/Tracer mP value is greater than 100 mP or negative, please refer to the user manual for your instrument and reset the G factor.
What is the best type of microplate reader for fluorescence polarization (FP) assays?

For FP assays, plate readers must specifically have FP built in. Please see our instrument compatibility portal (http://www.thermofisher.com/us/en/home/industrial/pharma-biopharma/drug-discovery-development/dd-misc/instrument-compatibility-portal.html).
How can I test the fluorescence polarization (FP) settings of my microplate reader before doing the assay?

We offer a FP One-Step Reference Kit (https://www.thermofisher.com/order/catalog/product/P3088) to detect in green (Ex/Em = 488/535 nm) and red (Ex/Em = 525/590 nm) emission ranges, suitable for the PolarScreen Green and Red Assays. The alternative is to perform the Controls in the respective assay kit prior to performing the actual assay using test compounds.

For Research Use Only. Not for use in diagnostic procedures.