| Assay Method Information | |
| | TR-FRET Assay |
| Description: | Akt2 inhibitory activity of compounds of the present invention was quantified employing the Akt2 TR-FRET assay as described in the following paragraphs.His-tagged human recombinant kinase full-length Akt2 expressed in insect cells and activated by PDK1 was purchased form Invitrogen (part number PV 3975). As substrate for the kinase reaction the biotinylated peptide biotin-Ahx- KKLNRTLSFAEPG (C-terminus in amide form) was used which can be purchased e.g. from the company Biosynthan GmbH (Berlinn-Buch, Germany). For the assay 50 nl of a 10Ofold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (Greiner Bio- One, Frickenhausen, Germany), 2 ul of a solution of Akt2 in assay buffer [50 mM TRIS/HCI pH 7.5, 5 mM MgCI2, 1 mM dithiothreitol, 0.02% (v/v) Triton X-100 (Sigma)] were added and the mixture was incubated for 15 min at 22C to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. |
| Affinity data for this assay | |
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