{"product_id":"ua-globret-intracellular-te-substateinhibitor-ua079048","title":"UA-Glo®BRET Intracellular TE Substate\/Inhibitor","description":"\u003ch4\u003eProduct Specification\u003c\/h4\u003e\u003cdiv class=\"responsive-table product-detail-table details-table\"\u003e\n\u003cbr\u003e\u003ctable style=\"width: 100%; height: auto;\"\u003e\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 22%;\"\u003e\u003cstrong\u003eSynonyms\u003c\/strong\u003e\u003c\/td\u003e\n\u003ctd style=\"width: 78%;\"\u003eBRET 细胞内TE底物和抑制剂\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 22%;\"\u003e\u003cstrong\u003eStability \u0026amp; Storage\u003c\/strong\u003e\u003c\/td\u003e\n\u003ctd style=\"width: 78%;\"\u003e\u003cp\u003eDry ice transportation. Store protected from light at -20°C or below, long-term storage (\u0026gt;3 months) at -80°C is recommended. Refer to the bottle label for expiration date.\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\u003c\/table\u003e\n\u003c\/div\u003e\u003ch4\u003eBackground\u003c\/h4\u003e\u003cdiv\u003e\u003cp\u003e\u003cspan\u003eUA-Glo® BRET Intracellular TE Substrates and Inhibitors include Youbio Biotech's proprietary micro-luciferase substrates and micro-luciferase inhibitors, which can be used to detect the binding (Target Engagement, TE) of micro-luciferase (NanoLuc®, NLuc) fused target proteins with compounds in cells. In the NanoBRET® TE experimental design, the NLuc-fused target protein serves as the energy donor for NanoBRET®, while the target protein-specific binding tracer acts as the energy acceptor. The light energy generated by the NLuc-catalyzed reaction of the micro-luciferase substrate from the NLuc-fused target protein can be absorbed by the tracer bound to the target protein nearby, and the light emitted by the excited tracer can be quantitatively detected. When a test compound binds to the target protein and competes with the tracer, the amount of tracer bound to the target protein decreases, resulting in a corresponding reduction in the emitted light energy. The strength of the tracer's emitted light can be quantitatively measured to calculate the degree of intracellular binding between the test compound and the target protein. Simultaneously detecting the NLuc luminescence value and calculating the ratio of the tracer to NLuc luminescence values can eliminate experimental errors and background interference, significantly improving detection sensitivity. The micro-luciferase inhibitor, added alongside the micro-luciferase substrate during detection, cannot enter cells but can inhibit the micro-luciferase activity of any extracellular NLuc-fused target proteins, further enhancing detection sensitivity.\u003c\/span\u003e\u003c\/p\u003e\u003c\/div\u003e\u003ch4\u003eComponents\u003c\/h4\u003e\u003cdiv\u003e\u003cp\u003eerror\u003c\/p\u003e\u003c\/div\u003e\u003ch4\u003eProtocol\u003c\/h4\u003e\u003cdiv\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eNanoBRET®\u003c\/span\u003e\u003cspan\u003e experiments require simultaneous detection of the luminescence intensity of the energy donor \u003c\/span\u003e\u003cspan\u003eNanoLuc®\u003c\/span\u003e\u003cspan\u003e and the energy acceptor. The peak emission wavelength of \u003c\/span\u003e\u003cspan\u003eNanoLuc®\u003c\/span\u003e\u003cspan\u003e is \u003c\/span\u003e\u003cspan\u003e460nm\u003c\/span\u003e\u003cspan\u003e, while the chromophore of the tracer (\u003c\/span\u003e\u003cspan\u003eNanoBRET®\u003c\/span\u003e\u003cspan\u003e \u003c\/span\u003e\u003cspan\u003e590 Dye\u003c\/span\u003e\u003cspan\u003e or \u003c\/span\u003e\u003cspan\u003e618 Dye\u003c\/span\u003e\u003cspan\u003e) emits at \u003c\/span\u003e\u003cspan\u003e590-610nm\u003c\/span\u003e\u003cspan\u003e. For detecting donor luminescence, a \u003c\/span\u003e\u003cspan\u003eBP\u003c\/span\u003e\u003cspan\u003e-type filter near \u003c\/span\u003e\u003cspan\u003e460nm\u003c\/span\u003e\u003cspan\u003e (bandwidth \u003c\/span\u003e\u003cspan\u003e8–80nm\u003c\/span\u003e\u003cspan\u003e) is recommended, such as \u003c\/span\u003e\u003cspan\u003eEm 450nm\/BP80\u003c\/span\u003e\u003cspan\u003e. For acceptor luminescence, an \u003c\/span\u003e\u003cspan\u003eLP\u003c\/span\u003e\u003cspan\u003e-type filter near \u003c\/span\u003e\u003cspan\u003e600-610nm\u003c\/span\u003e\u003cspan\u003e is suggested, such as \u003c\/span\u003e\u003cspan\u003eEm 610nm\/LP\u003c\/span\u003e\u003cspan\u003e. Suitable equipment includes multi-functional plate readers capable of \u003c\/span\u003e\u003cspan\u003eBRET\u003c\/span\u003e\u003cspan\u003e detection, such as \u003c\/span\u003e\u003cspan\u003ePerkinElmer EnVision®\u003c\/span\u003e\u003cspan\u003e, \u003c\/span\u003e\u003cspan\u003eBMG Labtech CLARIOstar®\u003c\/span\u003e\u003cspan\u003e, and \u003c\/span\u003e\u003cspan\u003ePromega GloMaxR Discover System\u003c\/span\u003e\u003cspan\u003e. Filter selection and instrument settings for \u003c\/span\u003e\u003cspan\u003eNanoBRET®\u003c\/span\u003e\u003cspan\u003e detection can be referenced from device manuals and relevant literature.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eThe following experiment demonstrates the application of \u003c\/span\u003e\u003cspan\u003eUA-Glo® BRET\u003c\/span\u003e\u003cspan\u003e intracellular \u003c\/span\u003e\u003cspan\u003eTE\u003c\/span\u003e\u003cspan\u003e substrates and inhibitors in \u003c\/span\u003e\u003cspan\u003eNanoBRET®\u003c\/span\u003e\u003cspan\u003e \u003c\/span\u003e\u003cspan\u003eTE\u003c\/span\u003e\u003cspan\u003e assays, using the measurement of compound \u003c\/span\u003e\u003cspan\u003eEC\u003c\/span\u003e\u003csub\u003e\u003cspan\u003e50\u003c\/span\u003e\u003c\/sub\u003e\u003cspan\u003e for \u003c\/span\u003e\u003cspan\u003eNLuc\u003c\/span\u003e\u003cspan\u003e-fused target proteins as an example. Detailed \u003c\/span\u003e\u003cspan\u003eHEK293\u003c\/span\u003e\u003cspan\u003e transfection methods, compound and tracer usage can be referenced from relevant literature and product manuals, such as \u003c\/span\u003e\u003cspan\u003ePromega\u003c\/span\u003e\u003cspan\u003e's \u003c\/span\u003e\u003cspan\u003eIntracellular TE Nano-Glo®\u003c\/span\u003e\u003cspan\u003e \u003c\/span\u003e\u003cspan\u003eSubstrate\/Inhibitor\u003c\/span\u003e\u003cspan\u003e, \u003c\/span\u003e\u003cspan\u003eIntracellular TE Nano-Glo®\u003c\/span\u003e\u003cspan\u003e \u003c\/span\u003e\u003cspan\u003eVivazine™\/Inhibitor\u003c\/span\u003e\u003cspan\u003e, and \u003c\/span\u003e\u003cspan\u003eNanoBRET® Target Engagement Intracellular Kinase Assay, Adherent Format\u003c\/span\u003e\u003cspan\u003e, or optimized by users based on specific experiments.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e1)\u003c\/span\u003e\u003cspan\u003eTransiently transfect \u003c\/span\u003e\u003cspan\u003eHEK293\u003c\/span\u003e\u003cspan\u003e cells with plasmids encoding \u003c\/span\u003e\u003cspan\u003eNLuc\u003c\/span\u003e\u003cspan\u003e-fused target proteins.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e2)\u003c\/span\u003e\u003cspan\u003eSeed transfected cells at an appropriate density in a 96-well clear-bottom white cell culture plate (\u003c\/span\u003e\u003cspan\u003e100μL\u003c\/span\u003e\u003cspan\u003e\/well). The recommended seeding medium is \u003c\/span\u003e\u003cspan\u003eOpti-MEM® I\u003c\/span\u003e\u003cspan\u003e low-serum medium (phenol red-free) \u003c\/span\u003e\u003cspan\u003e+1% FBS\u003c\/span\u003e\u003cspan\u003e.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e3)\u003c\/span\u003e\u003cspan\u003eIncubate the cell plate at \u003c\/span\u003e\u003cspan\u003e37℃\u003c\/span\u003e\u003cspan\u003e, \u003c\/span\u003e\u003cspan\u003e5% CO\u003c\/span\u003e\u003csub\u003e\u003cspan\u003e2\u003c\/span\u003e\u003c\/sub\u003e\u003cspan\u003e overnight (\u003c\/span\u003e\u003cspan\u003e20-30hr\u003c\/span\u003e\u003cspan\u003e).\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e4)\u003c\/span\u003e\u003cspan\u003ePrepare \u003c\/span\u003e\u003cspan\u003e20x\u003c\/span\u003e\u003cspan\u003e tracer: After \u003c\/span\u003e\u003cspan\u003e20-30hr\u003c\/span\u003e\u003cspan\u003e incubation, prepare the tracer at \u003c\/span\u003e\u003cspan\u003e20x\u003c\/span\u003e\u003cspan\u003e final concentration according to literature\/product manual recommendations or user optimization. First dilute the tracer in \u003c\/span\u003e\u003cspan\u003eDMSO\u003c\/span\u003e\u003cspan\u003e to \u003c\/span\u003e\u003cspan\u003e100x\u003c\/span\u003e\u003cspan\u003e, then further dilute to \u003c\/span\u003e\u003cspan\u003e20x\u003c\/span\u003e\u003cspan\u003e (\u003c\/span\u003e\u003cspan\u003e5% DMSO\u003c\/span\u003e\u003cspan\u003e) using tracer dilution buffer. Use polypropylene tubes to minimize wall adsorption.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e5)\u003c\/span\u003e\u003cspan\u003ePrepare test compounds: Serially dilute compounds in \u003c\/span\u003e\u003cspan\u003eDMSO\u003c\/span\u003e\u003cspan\u003e to \u003c\/span\u003e\u003cspan\u003e1000x\u003c\/span\u003e\u003cspan\u003e final concentration, then dilute to \u003c\/span\u003e\u003cspan\u003e10x\u003c\/span\u003e\u003cspan\u003e in \u003c\/span\u003e\u003cspan\u003eOpti-MEM® I\u003c\/span\u003e\u003cspan\u003e low-serum medium (phenol red-free).\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e6)\u003c\/span\u003e\u003cspan\u003eAdd \u003c\/span\u003e\u003cspan\u003e5μL\u003c\/span\u003e\u003cspan\u003e \u003c\/span\u003e\u003cspan\u003e20x\u003c\/span\u003e\u003cspan\u003e tracer and \u003c\/span\u003e\u003cspan\u003e10μL\u003c\/span\u003e\u003cspan\u003e \u003c\/span\u003e\u003cspan\u003e10x\u003c\/span\u003e\u003cspan\u003e test compound to each well. Include controls (solvent control, positive compound control, tracer-free solvent control, etc.). Mix by shaking for \u003c\/span\u003e\u003cspan\u003e15sec\u003c\/span\u003e\u003cspan\u003e.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e7)\u003c\/span\u003e\u003cspan\u003eIncubate at \u003c\/span\u003e\u003cspan\u003e37℃\u003c\/span\u003e\u003cspan\u003e, \u003c\/span\u003e\u003cspan\u003e5% CO\u003c\/span\u003e\u003csub\u003e\u003cspan\u003e2\u003c\/span\u003e\u003c\/sub\u003e\u003cspan\u003e for \u003c\/span\u003e\u003cspan\u003e2hr\u003c\/span\u003e\u003cspan\u003e or user-optimized duration.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e8)\u003c\/span\u003e\u003cspan\u003ePrepare \u003c\/span\u003e\u003cspan\u003e3x\u003c\/span\u003e\u003cspan\u003e intracellular \u003c\/span\u003e\u003cspan\u003eTE\u003c\/span\u003e\u003cspan\u003e detection reagent: Dilute the micro-luciferase substrate to \u003c\/span\u003e\u003cspan\u003e167x\u003c\/span\u003e\u003cspan\u003e and the micro-luciferase inhibitor to \u003c\/span\u003e\u003cspan\u003e500x\u003c\/span\u003e\u003cspan\u003e in \u003c\/span\u003e\u003cspan\u003eOpti-MEM® I\u003c\/span\u003e\u003cspan\u003e low-serum medium (phenol red-free). Mix gently by inverting \u003c\/span\u003e\u003cspan\u003e5-10\u003c\/span\u003e\u003cspan\u003e times. Prepare fresh and discard unused reagent.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e9)\u003c\/span\u003e\u003cspan\u003eRemove the cell plate and equilibrate to room temperature.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e10)\u003c\/span\u003e\u003cspan\u003eAdd \u003c\/span\u003e\u003cspan\u003e50μL\u003c\/span\u003e\u003cspan\u003e \u003c\/span\u003e\u003cspan\u003e3x\u003c\/span\u003e\u003cspan\u003e intracellular \u003c\/span\u003e\u003cspan\u003eTE\u003c\/span\u003e\u003cspan\u003e detection reagent to each well. Incubate at room temperature for \u003c\/span\u003e\u003cspan\u003e2min\u003c\/span\u003e\u003cspan\u003e.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e11)\u003c\/span\u003e\u003cspan\u003eMeasure donor (e.g., \u003c\/span\u003e\u003cspan\u003e460nm\u003c\/span\u003e\u003cspan\u003e) and acceptor (e.g., \u003c\/span\u003e\u003cspan\u003e610nm\u003c\/span\u003e\u003cspan\u003e) luminescence signals using a \u003c\/span\u003e\u003cspan\u003eNanoBRET\u003c\/span\u003e\u003csup\u003e\u003cspan\u003eTM\u003c\/span\u003e\u003c\/sup\u003e\u003cspan\u003e-compatible multi-functional plate reader. Read within \u003c\/span\u003e\u003cspan\u003e10min\u003c\/span\u003e\u003cspan\u003e after adding detection reagent.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e12)\u003c\/span\u003e\u003cspan\u003eData processing: The ratio of acceptor (\u003c\/span\u003e\u003cspan\u003e610nm\u003c\/span\u003e\u003cspan\u003e) to donor (\u003c\/span\u003e\u003cspan\u003e460nm\u003c\/span\u003e\u003cspan\u003e) signals (\u003c\/span\u003e\u003cspan\u003eBU\u003c\/span\u003e\u003cspan\u003e, multiplied by \u003c\/span\u003e\u003cspan\u003e1000\u003c\/span\u003e\u003cspan\u003e for \u003c\/span\u003e\u003cspan\u003emBU\u003c\/span\u003e\u003cspan\u003e) represents the \u003c\/span\u003e\u003cspan\u003eNanoBRET\u003c\/span\u003e\u003csup\u003e\u003cspan\u003eTM\u003c\/span\u003e\u003c\/sup\u003e\u003cspan\u003e ratio. Calculate the corrected \u003c\/span\u003e\u003cspan\u003eNanoBRET\u003c\/span\u003e\u003csup\u003e\u003cspan\u003eTM\u003c\/span\u003e\u003c\/sup\u003e\u003cspan\u003e ratio by subtracting the tracer-free control ratio from the sample ratio.\u003c\/span\u003e\u003c\/p\u003e\n\u003c\/div\u003e\u003ch4\u003eGuidelines\u003c\/h4\u003e\u003cdiv\u003e\u003cp style=\"margin-bottom: 0px;\"\u003e1) Different batches are not recommended for mixed use  2) Do not alter the amount of detection reagents without rigorous validation  3) For research use only  4) NanoLuc and NanoBRET are registered trademarks of Promega Corporation\u003c\/p\u003e\u003c\/div\u003e","brand":"UA BIOSCIENCE","offers":[{"title":"100assays","offer_id":43095508418635,"sku":"UA079048-100assays","price":270.0,"currency_code":"USD","in_stock":true},{"title":"1000assays","offer_id":43095508451403,"sku":"UA079048-1000assays","price":1370.0,"currency_code":"USD","in_stock":true},{"title":"10000assays","offer_id":43095508484171,"sku":"UA079048-10000assays","price":8705.0,"currency_code":"USD","in_stock":true}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/0590\/8375\/1499\/files\/1710709f-81bb-418c-9e10-db792db2c2ff.png?v=1788217381","url":"https:\/\/www.antbioinc.com\/products\/ua-globret-intracellular-te-substateinhibitor-ua079048","provider":"AntBio","version":"1.0","type":"link"}