| Assay Method Information | |
| | TR-FRET Assay |
| Description: | Kinase reaction conditions:400 nM GFP-4E-BP1, 8 uM ATP, -150 ng/mL mTOR, 50 mM HEPES pH 7.5, 0.01% Polysorbate 20, 1 mM EGTA, 10 mM MnC12, and variable amounts of test compounds.Preparation of Reagents:Note: Thaw and keep mTOR, the substrate, ATP, and the antibody on ice prior to making working dilutions. Working dilutions of these components can be kept at room temperature for short periods of time the day of use.1. Add 2 ml of 5X Assay Buffer to 8 ml water to prepare 10 ml of IX Assay Buffer. Note: The concentration of IX Assay Buffer is 50 mM HEPES pH 7.5, 0.01% Polysorbate 20, 1 mM EGTA, and 10 mM MnC12.2. Prepare Antibody/EDTA Solution by first adding 2.75 ul of Tb-anti p4E-BPl Antibody to 2397 ul of LanthaScreenTM TR-FRET Dilution Buffer. Then, add 100 ul of 0.5 M EDTA. 3. Prepare 4X Substrate/Enzyme Solution by first adding 72 ul of GFP-4E-BP1 (22 uM) to 926 ul of IX Assay Buffer. Then, add 1.6 ul of mTOR (0.45 mg/mL). 4. |
| Affinity data for this assay | |
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