{"product_id":"ua-glo-kinase-adp-assay-with-pi3k-substrate-pip2-3ps-ua079043","title":"UA-Glo® Kinase ADP Assay with PI3K Substrate PIP2:3PS","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. Upon receipt of the kit, PIP2:3PS 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. ATP removal reagent and ADP detection reagent should be stored at -20°C or below protected from light. For long-term storage (\u0026gt;3 months), it is recommended to store at -80°C. After first use, aliquot and store at -20°C or below protected from light. ATP removal reagent and ADP detection reagent show no loss in signal intensity or function after 7 freeze-thaw cycles. They also maintain signal intensity and function when stored at room temperature (22°C) for 24 hours or at 4°C for 7 days. 5x PI3K kinase reaction buffer, 10x PIP2:3PS dilution buffer, and MgCl2 should be stored at -20°C or below protected from light. Refer to the reagent bottle labels for expiration dates.\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\u003ePhosphatidylinositol (4,5)-bisphosphate (PIP2) and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) are derivatives of phosphatidylinositol (PI). PIP2 and PIP3 participate in various cellular physiological processes through dynamic phosphorylation\/dephosphorylation. Phosphoinositide 3-kinase (PI3K) is a key enzyme that mediates signaling by phosphorylating phosphatidylinositol derivatives, and its activated PI3K\/Akt signaling pathway plays a central role in cell proliferation, metabolic regulation, and immune responses. A class of phosphoinositide 3-kinases (PI3K Class I) generates PIP3 by phosphorylating PIP2, which can participate in downstream cascades mediated by growth factor receptors. Abnormalities in this signaling pathway are highly correlated with mechanisms of tumor development.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp\u003e\u003cspan\u003eThe UA-Glo® Kinase ADP Detection Assay quantitatively measures kinase activity in enzymatic assays. This assay determines kinase activity by quantifying the amount of ADP, a product of the kinase reaction: the amount of ADP is directly proportional to kinase activity. The UA-Glo® Kinase ADP Detection Assay can be used with ATP concentrations up to 1 mM in kinase reactions. This 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 concentrations. 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 UA-Glo® Kinase ADP Detection Kit (with PI3K substrate PIP2) includes the kinase ADP detection reagent and optimized PI3K substrate PIP2:3PS, making it suitable for high-throughput screening of PI3K (Class I) 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\u003eUA-Glo® \u003c\/span\u003e\u003cspan\u003eKinase\u003c\/span\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e Assay Kit\u003c\/span\u003e\u003cspan\u003e \u003c\/span\u003e\u003cspan\u003e(\u003c\/span\u003e\u003cspan\u003eContaining\u003c\/span\u003e\u003cspan\u003ePI3K\u003c\/span\u003e\u003cspan\u003eSubstrate\u003c\/span\u003e\u003cspan\u003ePIP2:3PS)\u003c\/span\u003e\u003cspan\u003e \u003c\/span\u003e\u003cspan\u003eComponents and specifications are as follows.\u003c\/span\u003e\u003cspan\u003e Detectable\u003c\/span\u003e\u003cspan\u003e96-\/384-\u003c\/span\u003e\u003cspan\u003ewell plate reaction count per well\u003c\/span\u003e\u003cspan\u003e25μL \/ 5μL\u003c\/span\u003e\u003cspan\u003e kinase reaction calculation.\u003c\/span\u003e\u003c\/p\u003e\n\u003ctable style=\"border-collapse:collapse;width:auto;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-weight:bold\"\u003eSpecification\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-weight:bold\"\u003eComponents\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-weight:bold\"\u003e96-well Plate\u003c\/span\u003e\u003cspan style=\"font-weight:bold\"\u003eReaction Count\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan style=\"font-weight:bold\"\u003e384-well Plate\u003c\/span\u003e\u003cspan style=\"font-weight:bold\"\u003eReaction Count\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e1,000 assays\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003ePIP2:3PS (1mg\/mL): 0.25mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e10x PIP2:3PS\u003c\/span\u003e\u003cspan\u003e Dilution Buffer\u003c\/span\u003e\u003cspan\u003e: 1mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eMgCl\u003c\/span\u003e\u003csub\u003e\u003cspan\u003e2\u003c\/span\u003e\u003c\/sub\u003e\u003cspan\u003e(1M): 1mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e5x PI3K\u003c\/span\u003e\u003cspan\u003e Kinase Reaction Buffer\u003c\/span\u003e\u003cspan\u003e: \u003c\/span\u003e\u003cspan\u003e1.5mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e Removal Reagent\u003c\/span\u003e\u003cspan\u003e: 5mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e Detection Reagent\u003c\/span\u003e\u003cspan\u003e: 10ml\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eATP (10mM):0.5mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eADP (10mM): 0.5mL\u003c\/span\u003e\u003c\/p\u003e\n\u003c\/td\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e200 \u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e1,000 \u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e10,000 assays\u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003ePIP2:3PS (1mg\/mL): 10x0.25mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e10x PIP2:3PS\u003c\/span\u003e\u003cspan\u003e Dilution Buffer\u003c\/span\u003e\u003cspan\u003e: 5x1mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eMgCl\u003c\/span\u003e\u003csub\u003e\u003cspan\u003e2\u003c\/span\u003e\u003c\/sub\u003e\u003cspan\u003e (1M): 1mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e5x PI3K\u003c\/span\u003e\u003cspan\u003e Kinase Reaction Buffer\u003c\/span\u003e\u003cspan\u003e:\u003c\/span\u003e\u003cspan\u003e 15mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eATP\u003c\/span\u003e\u003cspan\u003e Removal Reagent\u003c\/span\u003e\u003cspan\u003e: 50mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eADP\u003c\/span\u003e\u003cspan\u003e Detection Reagent\u003c\/span\u003e\u003cspan\u003e: 100ml\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eATP (10mM): 5mL\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:center\"\u003e\u003cspan\u003eADP (10mM): 5mL\u003c\/span\u003e\u003c\/p\u003e\n\u003c\/td\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e2,000 \u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003ctd style=\"text-align:center;vertical-align:middle;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000;border:1px solid #000000\"\u003e\u003cp style=\"text-align:center\"\u003e\u003cspan\u003e10,000 \u003c\/span\u003e\u003c\/p\u003e\u003c\/td\u003e\n\u003c\/tr\u003e\n\u003c\/table\u003e\n\u003cbr\u003e\n\u003c\/div\u003e\u003ch4\u003eProtocol\u003c\/h4\u003e\u003cdiv\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eThe PI3K kinase reaction is performed in 96-well or 384-well white opaque assay plates. The recommended reaction volume is 25μL for 96-well plates and 5μL for 384-well plates. Test compounds with concentration gradients can be added to the kinase reaction. The PI3K kinase reaction is recommended to be carried out at room temperature (23°C-25°C) for 1 hour, or under customer-optimized temperature and time conditions. No additional reagents are required to terminate the kinase reaction after completion. If termination reagents are needed due to special experimental requirements, avoid using magnesium ion chelators such as EDTA. The UA-Glo® kinase ADP detection reaction requires magnesium ions.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eThe kit includes a 5x PI3K kinase reaction buffer. Users may also use their own optimized PI3K kinase reaction buffer and cofactors. It is recommended to prepare the PI3K kinase reaction buffer as a 5x stock solution.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eThe concentration of PI3K kinase in the reaction should be optimized according to the supplier's instructions. For experiments requiring an appropriate signal-to-noise ratio, the kinase concentration within the linear signal response range can be used.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan\u003eThe final concentration of PIP2:3PS substrate in the PI3K kinase reaction is 0.05mg\/mL. The recommended ATP concentration is 25μM or a user-optimized concentration. Some commercially available ATP contains residual ADP. Due to the high sensitivity of the UA-Glo® kinase ADP detection reagent, residual ADP in ATP may cause high background. Therefore, high-purity ATP should be used for the kinase reaction, such as the ATP provided in 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 style=\"font-weight:bold\"\u003eThe following experimental example demonstrates the testing of a PI3K inhibitor IC50, with a PI3K kinase reaction volume of 25μL.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"font-weight:bold\"\u003ePI3K 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 10x final concentration of 2x or 3x serially diluted test compounds. Add 2.5μL\/well of the diluted test compounds to a 96-well white (opaque bottom) assay plate. Include solvent control wells, positive compound control wells, and no-enzyme control wells. The final concentration of solvents such as DMSO in the kinase reaction should not exceed 1%.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e2)\u003c\/span\u003e\u003cspan\u003eThe PI3K kinase reaction buffer stock solution is 5x. Dilute it 1:1 with sterile ultrapure water to prepare a 2.5x solution as needed.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e3)\u003c\/span\u003e\u003cspan\u003eThe PIP2:3PS substrate stock solution is 20x (1mg\/mL). Dilute it to 2.5x PIP2:3PS substrate using the 10x PIP2:3PS dilution buffer as follows:\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003ea)\u003c\/span\u003e\u003cspan\u003eThaw the PIP2:3PS substrate stock solution and equilibrate to room temperature.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003eb)\u003c\/span\u003e\u003cspan\u003eVortex vigorously for at least 1 minute to mix.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003ec)\u003c\/span\u003e\u003cspan\u003eDilute to 2.5x PIP2:3PS substrate using the 10x PIP2:3PS dilution buffer. For example, add 125μL PIP2:3PS substrate to 250μL 10x PIP2:3PS dilution buffer, then add sterile ultrapure water to 1mL.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003ed)\u003c\/span\u003e\u003cspan\u003eVortex vigorously for at least 1 minute to mix.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e4)\u003c\/span\u003e\u003cspan\u003eMix the 2.5x PI3K kinase reaction buffer from step 2 and the 2.5x PIP2:3PS substrate from step 3 in a 1:1 ratio. This is the 1.25x PI3K kinase reaction buffer\/PIP2:3PS. Vortex to mix.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e5)\u003c\/span\u003e\u003cspan\u003eAdd PI3K to the 1.25x PI3K kinase reaction buffer\/PIP2:3PS at the optimized enzyme concentration. Pipette or invert to mix. This is the 1.25x PI3K kinase 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\u003eAdd 20μL\/well of the 1.25x PI3K kinase working solution to the 96-well plate prepared in step 1 (containing 2.5μL\/well of 10x final concentration serially diluted test compounds). For no-enzyme control wells, add 20μL\/well of 1.25x PI3K kinase reaction buffer\/PIP2:3PS.\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 room temperature for 10-20 minutes to allow binding of test compounds to PI3K.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e8)\u003c\/span\u003e\u003cspan\u003eInitiate the kinase reaction by adding ATP: Dilute the kit-provided 10mM ATP to 250μM with sterile ultrapure water. Add 2.5μL\/well of 250μM ATP to the 96-well plate from step 7. The final ATP concentration is 25μM, and the kinase reaction volume is 25μL.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e9)\u003c\/span\u003e\u003cspan\u003eShake the plate to mix 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 to 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 PI3K 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:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e1)\u003c\/span\u003e\u003cspan\u003eThaw the UA-Glo® kinase ATP removal reagent and equilibrate to room temperature. Invert to mix.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:left\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e2)\u003c\/span\u003e\u003cspan\u003eAdd MgCl2 to the UA-Glo® kinase ATP removal reagent to a final concentration of 10mM. Invert to mix.\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e3)\u003c\/span\u003e\u003cspan\u003eIf the kinase reaction was performed at non-room temperature (e.g., 30°C), equilibrate the plate to room temperature. The luciferase reaction in the ADP detection reagent is sensitive to temperature changes. Reagents and test samples\/plates must be equilibrated to room temperature, and the testing process should maintain a constant temperature (±1°C).\u003c\/span\u003e\u003c\/p\u003e\n\u003cp style=\"text-align:justify\"\u003e\u003cspan style=\"margin-right: 8px;\"\u003e4)\u003c\/span\u003e\u003cspan\u003eAdd 25 µL\/well of ATP removal reagent to the 96-well plate containing 25 µL\/well of kinase reaction. Shake to mix. 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;\"\u003e5)\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 UA-Glo® 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 50 µL\/well of ADP detection reagent to the 96-well plate after ATP removal. Shake to mix. 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\u003eMeasure the luminescence signal 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 stable signal, the plate can be read within 3 hours or longer 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 change the dosage of detection reagents  3) For research use only\u003c\/p\u003e\u003c\/div\u003e","brand":"UA BIOSCIENCE","offers":[{"title":"1,000assays","offer_id":43094137569355,"sku":"UA079043-1,000assays","price":1040.0,"currency_code":"USD","in_stock":true},{"title":"10,000assays","offer_id":43094137602123,"sku":"UA079043-10,000assays","price":8335.0,"currency_code":"USD","in_stock":true}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/0590\/8375\/1499\/files\/59e6269d-497a-4cc5-9b53-22399754ac4a.png?v=1788202813","url":"https:\/\/www.antbioinc.com\/products\/ua-glo-kinase-adp-assay-with-pi3k-substrate-pip2-3ps-ua079043","provider":"AntBio","version":"1.0","type":"link"}