| Assay Method Information | |
| | HTRF KRASG12C/SOS1 Binding Assay |
| Description: | The HTRF KRASG12C/SOS1 Binding Assay was used to measure the interaction between KRASG12C and SOS1 proteins, and evaluate the enzymatic activity of compounds against KRASG12C. The utilized proteins and assay reagents were obtained from the KRASG12C-SOS1 binding assay kits (Cisbio). Initially, a 2 mM stock solution of the test compound (dissolved in DMSO) was serially diluted in five-fold concentrations using DMSO to obtain eight working solutions 1 (200×). Subsequently, each of the eight working solutions 1 was further diluted in a 20-fold gradient by adding 5 μL into 95 μL of Diluent buffer, mixed completely with vortex mixer, resulting in eight working solutions 2 (10×). In a 384-well white flat-bottom plate, 4 μL of Tag2-KRASG12C solution, 2 μL of working solution 2 (10×) and 4 μL of Tag1-SOS1 solution were sequentially added to each well and mixed, followed by a 15-minute incubation at room temperature (RT). Subsequently, 5 μL of Anti-tag 1-Tb3+ stock solution and 5 μL of Anti-tag2-XL665 were added to each well, mixed, and incubated at 4° C. for 3 hours. Remove the plate and read on an HTRF® compatible reader, with the excitation light wavelength set at 337 nm and readings recorded at 620 nm and 665 nm. The data results were presented as the ratio of the 665 nm signal value to the 620 nm signal value for each well, calculated as follows: Ratio=104/(665 nm signal value)/(620 nm signal value). The inhibition rate was calculated using the following formula:%Inhibitionrate= [(Rationegative-Ratiocompound)/(Rationegative-RatioBlank)]×100Data were analyzed using GraphPad Prism with log (inhibitor) vs. response-Variable Slope (four parameters) fitting calculations for IC50 values. |
| Affinity data for this assay | |
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