{"product_id":"ua-gloactriia-enzyme-activity-inhibitor-screening-kit-ua079044","title":"UA-Glo®ActRIIA Enzyme Activity Inhibitor Screening Kit","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\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 shipping. Reagent expiration dates are indicated on the reagent bottle labels.\u003cbr\u003eUpon receipt of the kit, Substrate, ATP, and ADP should be stored at -80°C protected from light. After first use, aliquot as needed and store at -80°C protected from light to avoid repeated freeze-thaw cycles. The ActR IIA enzyme protein is sensitive to freeze-thaw cycles; after first use, aliquot into single-use portions and store at -80°C, with no more than 3 freeze-thaw cycles. The 1x Assay buffer can be stored at 4°C after first use or aliquoted and stored at -20°C as needed. The 1x Assay buffer D working solution with DTT should be prepared fresh before use.\u003cbr\u003eATP removal reagent and ADP detection reagent should be stored protected from light at -20°C or below; long-term storage (\u0026gt;3 months) is recommended at -80°C. After first use, aliquot as needed and store at -20°C or below protected from light. ATP removal reagent and ADP detection reagent show no loss in signal intensity or functionality after up to 7 freeze-thaw cycles, and no loss in signal intensity or functionality when stored at room temperature (22°C) for 24 hours or at 4°C for 7 days.\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\u003c\/tbody\u003e\u003c\/table\u003e\n\u003c\/div\u003e\u003ch4\u003eBackground\u003c\/h4\u003e\u003cdiv\u003e\n\u003cp\u003e\u003cspan\u003eActivin receptor type II (ActR IIA and IIB) is a transmembrane serine\/threonine kinase receptor belonging to the TGF-β superfamily of type II receptors. ActR IIA\/IIB serves as the primary receptor for activin and GDF11. Upon ligand binding, it recruits type I receptors, which phosphorylate and activate downstream SMAD2\/3 signaling. Additionally, this receptor mediates the MAPK and PI3K\/AKT signaling pathways.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003eIn Phase II clinical studies for obesity treatment, inhibition of the ActR II signaling pathway has been shown to significantly reduce fat while stimulating an increase in lean body mass (muscle). For obese patients, achieving substantial fat reduction while maintaining or increasing lean body mass is a critical need. Whether as a monotherapy or in combination with incretin (GLP-1)-based therapies, ActR II inhibition is the only clinically proven mechanism capable of achieving this beneficial overall body composition change.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003eThe Kinase ADP assay reagent quantitatively measures kinase activity in enzymatic experiments. This reagent determines kinase activity by quantifying the amount of ADP, a product of the kinase reaction: the amount of ADP is positively correlated with kinase activity. The ATP concentration in the kinase reaction can be up to 1 mM when using this reagent. The kit can detect most kinases, with substrates including peptides, proteins, lipids, or sugars. It is suitable for high-throughput homogeneous screening of kinase inhibitors and can distinguish between ATP-competitive and non-competitive inhibitors by adjusting ATP concentration. The kit can also be used to measure the activity of any ADP-producing enzyme, such as ATPase.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003eThe ActR IIA inhibitor screening kit includes ActR IIA enzyme protein, substrate, kinase reaction buffer (1x Assay buffer), and Kinase ADP assay reagent. It can be used to evaluate the effect of compounds on ActR IIA enzyme activity and is particularly suitable for high-throughput screening of ActR IIA inhibitors.\u003c\/span\u003e\u003c\/p\u003e\n\u003c\/div\u003e\u003ch4\u003eComponents\u003c\/h4\u003e\u003cdiv\u003e\n\u003cp style=\"text-align:justify;\"\u003e\u003cspan\u003eThe components and specifications of the ActR IIA inhibitor screening kit are as follows. The detectable number of reactions is calculated based on a 384-well plate with 5μL kinase reaction per well, and the reaction conditions are according to the example in the instructions.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left;\"\u003e\u003cspan\u003eSpecies: Human\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left;\"\u003e\u003cspan\u003eMolecular aliases: ACVR 2A, Activin receptor type IIA, ACTR IIA\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left;\"\u003e\u003cspan\u003eComponents and specifications:\u003c\/span\u003e\u003c\/p\u003e\n\u003cfigure class=\"table\"\u003e\u003ctable style=\"border-collapse:collapse;border:1px solid #000000;width:auto;\"\u003e\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003eComponent\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003eStorage condition\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e500T\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e1000T\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003eActR IIA (750 μg\/mL)\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e-80 °C\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e50μL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e50μL*2\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003eSubstrate (500 μM), 250X\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e-80 °C\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e12μL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e24μL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003eDTT (50 mM)\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e-20 °C\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e5μL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e10μL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e1X Assay buffer\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e-20 °C\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e5mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e10mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003eATP removal reagent\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e-20 °C\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e2.5mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e5mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003eADP detection reagent\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e-20 °C\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e5mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e10mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003eATP (10mM)\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e-80 °C\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e0.25mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e0.5mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003eADP (10mM)\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e-80 °C\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e0.25mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"border:1px solid #000000;text-align:center;vertical-align:middle;\"\u003e\u003cp style=\"text-align:center;\"\u003e\u003cspan\u003e0.5mL\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/tbody\u003e\u003c\/table\u003e\u003c\/figure\u003e\u003cp\u003e\u003cbr\u003e \u003c\/p\u003e\n\u003c\/div\u003e\u003ch4\u003eProtocol\u003c\/h4\u003e\u003cdiv\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eThe optimized reaction conditions for the ActR IIA kinase assay system include \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e1x Assay buffer\u003c\/span\u003e\u003cspan\u003e, \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003eActR IIA enzyme at a final concentration of 15 µg\/mL\u003c\/span\u003e\u003cspan\u003e, \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003esubstrate (Substrate) at a final concentration of 2 µM\u003c\/span\u003e\u003cspan\u003e, and \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003eATP at a final concentration of 10 µM\u003c\/span\u003e\u003cspan\u003e. Customers may further optimize these conditions as needed.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eThe ActR IIA kinase reaction is performed in 96-well or 384-well white opaque assay plates. A reaction volume of 25 µL is recommended for 96-well plates, and 5 µL for 384-well plates. A concentration gradient of test compounds may be added to the kinase reaction. The recommended temperature and duration for the kinase reaction are room temperature (23°C–25°C) for 1 hour, or as optimized by the customer. No additional reagents are required to terminate the kinase reaction. If termination is necessary for specific experimental requirements, avoid using magnesium ion chelators such as EDTA, as the kinase ADP detection reaction requires magnesium ions.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eSome commercially available ATP contains residual ADP. Due to the high sensitivity of the kinase ADP detection reagents, residual ADP in ATP may lead to high background signals. Therefore, high-purity ATP should be used for the kinase reaction, such as the ATP provided with the kit or other high-purity ATP, e.g., Sigma-Aldrich ATP (Cat# A2383, purity ≥99%) or higher-purity ATP.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eThe following experimental example demonstrates the testing of an ActR IIA inhibitor’s IC\u003csub\u003e50\u003c\/sub\u003e. The kinase reaction volume was 5 µL, with \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003eActR IIA enzyme at a final concentration of 15 µg\/mL\u003c\/span\u003e\u003cspan\u003e, \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003esubstrate (Substrate) at a final concentration of 2 µM\u003c\/span\u003e\u003cspan\u003e, and \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003eATP at a final concentration of 10 µM\u003c\/span\u003e\u003cspan\u003e, with DMSO not exceeding 1%. If users have liquid handling and dispensing equipment capable of handling very small volumes, it is recommended to prepare gradient-diluted test compounds directly in 100% DMSO during the compound preparation step. For example, 25 nL (200x test concentration) of compound can be added to a 384-well assay plate, and the volume and concentration of the subsequent kinase reaction working solution and substrate working solution should be adjusted accordingly (e.g., adding 4 µL of 1.25x kinase reaction solution and 1 µL of 5x substrate working solution), as long as the final concentrations in the kinase reaction remain consistent with the above.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003e \u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-weight:bold\"\u003eActR IIA Kinase Reaction\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e1)\u003c\/span\u003e\u003cspan\u003ePrepare 1x Assay buffer D working solution: Take the required amount of 1x Assay buffer and add DTT to a final concentration of 50 µM. This is the 1x Assay buffer D working solution, used for preparing subsequent compound, protein, and substrate working solutions. Prepare fresh for each experiment.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e2)\u003c\/span\u003e\u003cspan\u003eCompound preparation: Use the 1x Assay buffer D working solution to prepare 5x final concentration of 3x gradient-diluted test compounds. Note that the concentration of organic solvents (e.g., DMSO) should not exceed 3% in this step, ensuring that the DMSO concentration does not exceed 1% after adding the ActR IIA kinase reaction solution in step 5.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e3)\u003c\/span\u003e\u003cspan\u003eAdd 1 µL\/well of gradient-diluted test compounds to a 384-well white (opaque bottom) assay plate. Include solvent control wells, positive compound control wells, and no-enzyme control wells.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e4)\u003c\/span\u003e\u003cspan\u003ePrepare the 2.5x kinase reaction solution: Take the required amount of 1x Assay buffer D and add ActR IIA to a concentration of 37.5 µg\/mL (or as optimized by the customer). Mix gently by pipetting. This is the 2.5x kinase reaction solution.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e5)\u003c\/span\u003e\u003cspan\u003eAdd 2 µL\/well of the 2.5x ActR IIA kinase reaction solution to the 384-well assay plate prepared in step 3 (containing 1 µL of 5x gradient-diluted test compounds). For no-enzyme control wells, add 2 µL\/well of 1x Assay buffer D working solution.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e6)\u003c\/span\u003e\u003cspan\u003eIncubate at room temperature for 15 minutes to allow binding of the test compounds to the ActR IIA enzyme.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e7)\u003c\/span\u003e\u003cspan\u003ePrepare the 2.5x substrate working solution: Take the required amount of 1x Assay buffer D working solution, add Substrate to 5 µM and ATP to 25 µM, and mix by pipetting\/inverting. This is the 2.5x substrate working solution.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e8)\u003c\/span\u003e\u003cspan\u003eAdd 2 µL\/well of the 2.5x substrate working solution to the 384-well assay plate from step 6 to initiate the kinase reaction.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e9)\u003c\/span\u003e\u003cspan\u003eMix by shaking and incubate at room temperature (23°C–25°C) for 1 hour.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e10)\u003c\/span\u003e\u003cspan\u003eProceed with ADP detection as follows.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003e \u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-weight:bold\"\u003eADP Detection After ActR IIA Kinase Reaction\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e1.\u003c\/span\u003e\u003cspan style=\"font-weight:bold\"\u003eATP Removal After Kinase Reaction\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e1)\u003c\/span\u003e\u003cspan\u003eThaw the kinase ATP removal reagent and equilibrate to room temperature. Invert to mix. The luciferase reaction in the ADP detection reagent is sensitive to temperature changes. Reagents and test samples\/assay plates should be equilibrated to room temperature (23°C–25°C), and the temperature should remain constant (±1°C) during testing.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e2)\u003c\/span\u003e\u003cspan\u003eAdd 5 µL\/well of ATP removal reagent to the 384-well assay plate containing 5 µL\/well of kinase reaction. The volume ratio of kinase reaction, ATP removal reagent, and ADP detection reagent should be 1:1:2.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e3)\u003c\/span\u003e\u003cspan\u003eIncubate at room temperature for 40 minutes.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan\u003e \u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e2.\u003c\/span\u003e\u003cspan style=\"font-weight:bold\"\u003eADP Detection\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e1)\u003c\/span\u003e\u003cspan\u003eThaw the kinase ADP detection reagent and equilibrate to room temperature. Invert to mix. The ADP detection reagent may have slight precipitation after thawing; the supernatant can be used directly or centrifuged to remove precipitation.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e2)\u003c\/span\u003e\u003cspan\u003eAdd 10 µL\/well of ADP detection reagent to the 384-well assay plate after ATP removal. Mix by shaking and incubate in the dark at room temperature for 40 minutes.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e3)\u003c\/span\u003e\u003cspan\u003eRead luminescence signals using a luminometer or multifunctional plate reader. The signal can be read as early as 30 minutes after adding the ADP detection reagent. Due to the stability of the signal, readings can be taken up to 3 hours or longer after adding the detection reagent if needed.\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) Without strict verification, it is not recommended to change the dosage of detection reagents  3) For research use only\u003c\/p\u003e\u003c\/div\u003e","brand":"UA BIOSCIENCE","offers":[{"title":"500T","offer_id":43094330900555,"sku":"UA079044-500T","price":1490.0,"currency_code":"USD","in_stock":true},{"title":"1000T","offer_id":43094330933323,"sku":"UA079044-1000T","price":2500.0,"currency_code":"USD","in_stock":true}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/0590\/8375\/1499\/files\/777778c0-0e26-4402-bd42-d447a6644a60.png?v=1788206450","url":"https:\/\/www.antbioinc.com\/products\/ua-gloactriia-enzyme-activity-inhibitor-screening-kit-ua079044","provider":"AntBio","version":"1.0","type":"link"}