PolarScreen™ ER β競合アッセイキットは、潜在的なエストロゲン受容体(ER)βリガンドのハイスループットな蛍光偏光ベースのスクリーニングを高感度で効率的に行えます。このキットは、昆虫細胞で発現した全長のネイティブ(タグなし)ヒトER β(A15664)と、新規の高親和性蛍光エストロゲンリガンド(Fluormone™ ES2)を均質なミックスアンドリードアッセイ形式で使用します。このキットでは緑色の読み取り値を生成しますが、「赤色」のキットもあります(A15891)。どちらのキットも競合化合物のIC50を適切に測定します。色の選択は、スクリーニングする化合物のスペクトル特性と利用可能な機器によって異なります。詳細情報またはサポートについては、drugdiscoverytech@lifetech.comまでメールでお問い合わせください。
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.