| Assay Method Information | |
| | Biochemical Assay 1 |
| Description: | The expression and purification of N-terminal GST-tagged human K-RasG120 and N-terminal His-tagged human SOS1 is described below. Concentrations of protein batches used were optimized to be within the linear range of the HTRF signal. A Ras working solution was prepared in assay buffer containing typically 10 nM GST-hK-RasG12C and 2 nM antiGST-Eu(K) (Cisbio, France). A SOS working solution was prepared in assay buffer containing typically 20 nM His-hSOS1 and 10 nM anti-6His-XL665 (Cisbio, France). An inhibitor control solution was prepared in assay buffer containing 10 nM anti-6His-XL665 without hSOS1.Fifty nl of a 100-fold concentrated solution of the test compound in DMSO were transferred into a black microtiter test plate (384 or 1536, Greiner Bio-One, Germany). For this, either a Hummingbird liquid handler (Digilab, MA, USA) or an Echo acoustic system (Labcyte, CA, USA) was used.All steps of the assay were performed at 20° C. A volume of 2.5 μl of the Ras working solution was added to all wells of the test plate using a Multidrop dispenser (Thermo Labsystems). After 2 min preincubation, 2.5 μl of the SOS working solution were added to all wells except for those wells at the side of the test plate that were subsequently filled with 2.5 μl of the inhibitor control solution. After 60 min incubation the fluorescence was measured with a Pherastar (BMG, Germany) using the HTRF module (excitation 337 nm, emission 1: 620 nm, emission 2: 665 nm). |
| Affinity data for this assay | |
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