{"product_id":"ua-gloactriib-enzyme-activity-inhibitor-screening-kit-ua079045","title":"UA-Glo®ActRIIB 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 reagent kit, Substrate, ATP, and ADP should be stored at -80°C protected from light. After initial use, aliquot as needed and store at -80°C protected from light, avoiding repeated freeze-thaw cycles. The ActR IIB enzyme protein is sensitive to freeze-thaw cycles. After initial use, aliquot into single-use portions and store at -80°C, with freeze-thaw cycles not exceeding 3 times. The 1x Assay buffer can be stored at 4°C after initial use or aliquoted and stored at -20°C as needed. The 1x Assay buffer D working solution with DTT should be prepared fresh for immediate use.\u003cbr\u003eATP removal reagent and ADP detection reagent should be stored protected from light at -20°C or below. For long-term storage (\u0026gt;3 months), it is recommended to store at -80°C. After initial use, aliquot as needed and store at -20°C or below protected from light. The 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, inhibiting the ActRII 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, ActRII inhibition is clinically the only mechanism proven to achieve this beneficial overall body composition change.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003eThe Kinase ADP Assay Kit quantitatively measures kinase activity in enzymatic experiments. This kit 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 kit. It can detect most kinases, with substrates including peptides, proteins, lipids, or sugars. The kit is suitable for high-throughput homogeneous screening of kinase inhibitors and can distinguish between ATP-competitive and non-competitive inhibitors by adjusting ATP concentration. It can also be used to measure the activity of any enzyme that produces ADP, such as ATPase.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003eThe ActR IIB Inhibitor Screening Kit includes ActR IIB enzyme protein, substrate, kinase reaction buffer (1x Assay buffer), and Kinase ADP Assay Reagent. It can be used to assess the effect of compounds on ActR IIB enzyme activity and is particularly suitable for high-throughput screening of ActR IIB 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 IIB 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, under the conditions specified in the manual.\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 alias: ACVR 2B, Activin receptor type IIB, ACTR IIB\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 IIB (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 system for the \u003c\/span\u003e\u003cspan\u003eActR IIB\u003c\/span\u003e\u003cspan\u003e kinase reaction includes \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e1x Assay buffer\u003c\/span\u003e\u003cspan\u003e, \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003eActR IIB\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e enzyme at a final concentration of \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e15 µg\/mL\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e, substrate \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e(Substrate)\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e at a final concentration of \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e2 µM\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e, and \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003eATP\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e at a final concentration of \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e10 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 \u003c\/span\u003e\u003cspan\u003eActR IIB\u003c\/span\u003e\u003cspan\u003e kinase reaction is performed in 96-well or 384-well white opaque assay plates. A reaction volume of \u003c\/span\u003e\u003cspan\u003e25 µL\u003c\/span\u003e\u003cspan\u003e is recommended for 96-well plates, and \u003c\/span\u003e\u003cspan\u003e5 µL\u003c\/span\u003e\u003cspan\u003e for 384-well plates. Gradient concentrations of test compounds may be added to the kinase reaction. The reaction is typically conducted at room temperature (23°C–25°C) for 1 hour, or under conditions 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 \u003c\/span\u003e\u003cspan\u003eEDTA\u003c\/span\u003e\u003cspan\u003e, as the kinase \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e detection reaction requires magnesium ions.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eSome commercially available \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e contains residual \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e. Due to the high sensitivity of the kinase \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e detection reagents, residual \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e in \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e can lead to high background signals. Therefore, high-purity \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e should be used for the kinase reaction, such as the \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e provided with the kit or other high-purity \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e sources (e.g., Sigma-Aldrich \u003c\/span\u003e\u003cspan\u003eATP, Cat# A2383, purity ≥99%\u003c\/span\u003e\u003cspan\u003e).\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eThe following example demonstrates the measurement of \u003c\/span\u003e\u003cspan\u003eIC\u003c\/span\u003e\u003csub\u003e\u003cspan\u003e50\u003c\/span\u003e\u003c\/sub\u003e\u003cspan\u003e for an \u003c\/span\u003e\u003cspan\u003eActR IIB\u003c\/span\u003e\u003cspan\u003e inhibitor. The kinase reaction volume was \u003c\/span\u003e\u003cspan\u003e5 µL\u003c\/span\u003e\u003cspan\u003e, with final concentrations of \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003eActR IIB\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e enzyme at \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e15 µg\/mL\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e, substrate \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e(Substrate)\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e at \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e2 µM\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e, and \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003eATP\u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e at \u003c\/span\u003e\u003cspan style=\"color:#EE0000\"\u003e10 µM\u003c\/span\u003e\u003cspan\u003e. The final concentration of \u003c\/span\u003e\u003cspan\u003eDMSO\u003c\/span\u003e\u003cspan\u003e should not exceed \u003c\/span\u003e\u003cspan\u003e1%\u003c\/span\u003e\u003cspan\u003e. If the user has liquid handling equipment capable of dispensing very small volumes, it is recommended to prepare gradient-diluted test compounds directly in \u003c\/span\u003e\u003cspan\u003e100% DMSO\u003c\/span\u003e\u003cspan\u003e (e.g., \u003c\/span\u003e\u003cspan\u003e25 nL\u003c\/span\u003e\u003cspan\u003e of a 200x test concentration) into the 384-well assay plate. The volume and concentration of the subsequent kinase reaction working solution and substrate working solution should be adjusted accordingly (e.g., adding \u003c\/span\u003e\u003cspan\u003e4 µL\u003c\/span\u003e\u003cspan\u003e of 1.25x kinase reaction solution and \u003c\/span\u003e\u003cspan\u003e1 µL\u003c\/span\u003e\u003cspan\u003e of 5x substrate working solution), as long as the final concentrations match the above conditions.\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 IIB 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: Add \u003c\/span\u003e\u003cspan\u003eDTT\u003c\/span\u003e\u003cspan\u003e to the required volume of \u003c\/span\u003e\u003cspan\u003e1x Assay buffer\u003c\/span\u003e\u003cspan\u003e to a final concentration of \u003c\/span\u003e\u003cspan\u003e50 µM\u003c\/span\u003e\u003cspan\u003e. This is the \u003c\/span\u003e\u003cspan\u003e1x Assay buffer D\u003c\/span\u003e\u003cspan\u003e 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 \u003c\/span\u003e\u003cspan\u003e1x Assay buffer D\u003c\/span\u003e\u003cspan\u003e working solution to prepare 5x final concentration of 3x gradient-diluted test compounds. Note that the concentration of organic solvents (e.g., \u003c\/span\u003e\u003cspan\u003eDMSO\u003c\/span\u003e\u003cspan\u003e) should not exceed \u003c\/span\u003e\u003cspan\u003e3%\u003c\/span\u003e\u003cspan\u003e at this step, ensuring that the final \u003c\/span\u003e\u003cspan\u003eDMSO\u003c\/span\u003e\u003cspan\u003e concentration does not exceed \u003c\/span\u003e\u003cspan\u003e1%\u003c\/span\u003e\u003cspan\u003e after adding the \u003c\/span\u003e\u003cspan\u003eActR IIB\u003c\/span\u003e\u003cspan\u003e 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 \u003c\/span\u003e\u003cspan\u003e1 µL\/well\u003c\/span\u003e\u003cspan\u003e 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 2.5x kinase reaction solution: Add \u003c\/span\u003e\u003cspan\u003eActR IIB\u003c\/span\u003e\u003cspan\u003e to the required volume of \u003c\/span\u003e\u003cspan\u003e1x Assay buffer D\u003c\/span\u003e\u003cspan\u003e to a final concentration of \u003c\/span\u003e\u003cspan\u003e37.5 µg\/mL\u003c\/span\u003e\u003cspan\u003e (or customer-optimized enzyme concentration). 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 \u003c\/span\u003e\u003cspan\u003e2 µL\/well\u003c\/span\u003e\u003cspan\u003e of the 2.5x \u003c\/span\u003e\u003cspan\u003eActR IIB\u003c\/span\u003e\u003cspan\u003e kinase reaction solution to the 384-well plate prepared in step 3 (containing 1 µL of 5x gradient-diluted test compounds). For no-enzyme control wells, add \u003c\/span\u003e\u003cspan\u003e2 µL\/well\u003c\/span\u003e\u003cspan\u003e of \u003c\/span\u003e\u003cspan\u003e1x Assay buffer D\u003c\/span\u003e\u003cspan\u003e 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 \u003c\/span\u003e\u003cspan\u003e15 minutes\u003c\/span\u003e\u003cspan\u003e to allow binding of the test compounds to the \u003c\/span\u003e\u003cspan\u003eActR IIB\u003c\/span\u003e\u003cspan\u003e 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 2.5x substrate working solution: Add \u003c\/span\u003e\u003cspan\u003eSubstrate\u003c\/span\u003e\u003cspan\u003e to \u003c\/span\u003e\u003cspan\u003e5 µM\u003c\/span\u003e\u003cspan\u003e and \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e to \u003c\/span\u003e\u003cspan\u003e25 µM\u003c\/span\u003e\u003cspan\u003e in the required volume of \u003c\/span\u003e\u003cspan\u003e1x Assay buffer D\u003c\/span\u003e\u003cspan\u003e working solution. Mix by pipetting or inversion. 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 \u003c\/span\u003e\u003cspan\u003e2 µL\/well\u003c\/span\u003e\u003cspan\u003e of the 2.5x substrate working solution to the 384-well 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 plate shaking and incubate at room temperature (23°C–25°C) for \u003c\/span\u003e\u003cspan\u003e1 hour\u003c\/span\u003e\u003cspan\u003e.\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 \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e 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 IIB 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 \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e removal reagent and equilibrate to room temperature. Mix by inversion. The luciferase reaction in the \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e detection reagent is sensitive to temperature changes. Reagents and test samples\/assay plates must be equilibrated to room temperature (23°C–25°C), and the temperature should remain stable (±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 \u003c\/span\u003e\u003cspan\u003e5 µL\/well\u003c\/span\u003e\u003cspan\u003e of \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e removal reagent to the 384-well plate containing the \u003c\/span\u003e\u003cspan\u003e5 µL\/well\u003c\/span\u003e\u003cspan\u003e kinase reaction. Mix by shaking. The volume ratio of kinase reaction, \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e removal reagent, and \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e 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 \u003c\/span\u003e\u003cspan\u003e40 minutes\u003c\/span\u003e\u003cspan\u003e.\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 \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e detection reagent and equilibrate to room temperature. Mix by inversion. If precipitation occurs after thawing, use the supernatant directly or centrifuge to remove the precipitate.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e2)\u003c\/span\u003e\u003cspan\u003eAdd \u003c\/span\u003e\u003cspan\u003e10 µL\/well\u003c\/span\u003e\u003cspan\u003e of \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e detection reagent to the 384-well plate after \u003c\/span\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e removal. Mix by shaking and incubate in the dark at room temperature for \u003c\/span\u003e\u003cspan\u003e40 minutes\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\u003eMeasure luminescence signals using a luminometer or multifunctional plate reader. The signal can be read as early as \u003c\/span\u003e\u003cspan\u003e30 minutes\u003c\/span\u003e\u003cspan\u003e after adding the \u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e detection reagent. Due to signal stability, readings can be taken up to \u003c\/span\u003e\u003cspan\u003e3 hours\u003c\/span\u003e\u003cspan\u003e 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 validation, it is not recommended to alter the dosage of detection reagents  3) For research use only\u003c\/p\u003e\u003c\/div\u003e","brand":"UA BIOSCIENCE","offers":[{"title":"500T","offer_id":43094330835019,"sku":"UA079045-500T","price":1490.0,"currency_code":"USD","in_stock":true},{"title":"1000T","offer_id":43094330867787,"sku":"UA079045-1000T","price":2500.0,"currency_code":"USD","in_stock":true}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/0590\/8375\/1499\/files\/ce9661ea-138e-4d21-8050-d0a9a4828888.png?v=1788206448","url":"https:\/\/www.antbioinc.com\/products\/ua-gloactriib-enzyme-activity-inhibitor-screening-kit-ua079045","provider":"AntBio","version":"1.0","type":"link"}