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| | Kinase Activity Assay |
| Description: | The mTOR kinase TR-FRET assay utilizes a physiologically relevant protein substrate for mTOR (4E-BP1, labeled with an acceptor fluorophore (Green Fluorescent Protein) and paired with a corresponding Tb-labeled phospho-specific antibody. The assay itself is divided into two phases: the reaction phase and the detection phase. In the reaction phase, all components required for the kinase reaction are added to the well, including the labeled protein substrate. The reaction is allowed to incubate for 60 minutes. After the reaction, EDTA is added to stop the kinase reaction, and terbium-labeled antibody is added to bind phosphorylated product. Because the terbium chelate is stable at the EDTA concentrations used to stop a kinase assay, the antibody and EDTA can be pre-mixed prior to addition to minimize pipetting steps. Binding of the terbium labeled antibody to the fluorophore-labeled phosphorylated product brings the terbium and GFP into proximity, resulting in an increase in TR-FRET. |
| Affinity data for this assay | |
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