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Invitrogen™ PolarScreen™ PPARγ-Competitor Assay Kit, Green

Catalog No. PV6136
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800 x 20 μL
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PV6136 800 x 20 μL
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Catalog No. PV6136 Supplier Invitrogen™ Supplier No. PV6136
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Description

PolarScreen™ PPARγ-Competitor Assay Kit, Green

The PPARγ (Peroxisome Proliferator-Activated Receptor-gamma) Competitor Assay, Green uses the human-derived recombinant PPARγ ligand-binding domain (PPARγ-LBD) tagged with an N-terminal GST-tag and a novel, tight-binding, selective fluorescent PPARγ ligand (Fluormone™ PPARγ Green) in a homogenous, mix-and-read assay format. PPARγ-LBD/Fluormone™ PPARγ Green complex produces high polarization. This complex is then added to individual test compounds in microwell plates. Competitors displace the fluorescent Fluormone™ PPARγ Green Ligand from the PPARγ-LBD/Fluormone™ PPARγ Green complex, causing the fluorescent ligand to tumble rapidly during its fluorescence lifetime, resulting in a low polarization value. Noncompetitors will not displace the fluorescent ligand from the complex, so the polarization value remains high. The shift in polarization value in the presence of the test compounds is used to determine relative affinity of test compounds for the PPARγ-LBD.

Specifications

Color Green
Detection Method Fluorescence
For Use With (Equipment) Microplate Reader
Quantity 800 x 20 μL
Packaging 384-well Plate
Content And Storage PV3356 — Fluormone™ PPARγ Green (-20°C)
PV4546 — PPARγ-LBD (-80°C)
PV3358 — PPARγ Green Screening Buffer (room temperature)
P2325 — 1M DTT (-80°C or -20°C)
Assay Entry Biochemical competitive binding
Excitation/Emission 485/535
Gene ID (Entrez) 5468
Readout End Point
Shipping Condition Dry Ice
Target Entry PPARG, PPAR gamma, NR1C3
Product Line PolarScreen
For Use With (Application) Competitive Binding Assay, Fluorescence Polarization
Ligand PPAR
Product Type Assay Kit
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Frequently Asked Questions (FAQs)

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.