| Assay Method Information | |
| | Intracellular Axl Phosphorylation Inhibitory Activity |
| Description: | A phosphorylated Axl (hereinafter, referred to as pAxl) inhibition test was conducted using a human non-small cell lung cancer-derived cell line NCI-H1299.The NCI-H1299 cells were suspended in a medium (RPMI1640 medium containing 10% fetal bovine serum), then inoculated at 15000 cells/100 μl/well to each 96-well multiwall plate, and cultured at 37° C. for 1 day in the presence of 5% CO2. On the next day, the medium was discarded, and a medium was added at 100 μl/well to the plate, followed by culture at 37° C. for 1 day in the presence of 5% CO2. Each test compound was dissolved in DMSO and diluted with an FBS-free medium to prepare a sample solution (DMSO concentration: 2%). A medium or a medium supplemented with the sample was added at 25 μl/well (DMSO concentration: 0.4%) to the plate and incubated at 37° C. for 1 hour in the presence of 5% CO2.GAS6 (R&D Systems Inc., model: 885-GS) was diluted into 6 μg/ml with an FBS-free medium, then added at 25 μl/well to the plate, and incubated at 37° C. for 10 minutes in the presence of 5% CO2 after stirring.The supernatant was discarded, and a solution of a 37% formalin solution diluted to 4% with phosphate-buffered saline (PBS) (hereinafter, a 4% formalin solution) was added at 0.1 ml/well to the plate, which was then left standing at room temperature for 10 minutes. Next, the 4% formalin solution was discarded, and a solution of Triton X-100 diluted to 0.1% with PBS (hereinafter, referred to as a wash buffer) was added at 0.2 ml/well to the plate and discarded by decantation. An excess of water was removed on paper towel.Subsequently, 10% NaN3 and 110 μl of H2O2 were added to 10.7 mL of a wash buffer (hereinafter, referred to as a quenching buffer), and this quenching buffer was added at 0.1 ml/well to the plate, which was then left standing at room temperature for 15 minutes.The quenching buffer was discarded, and a wash buffer was added at 0.2 ml/well to the plate and discarded by decantation. An excess of water was removed on a paper towel. Skimmed milk (WAKO #198-10605) was added (final concentration 5%) to a wash buffer (hereinafter, referred to as a blocking buffer), and this blocking buffer was added at 0.25 ml/well to the plate, which was then left standing at room temperature for 1 hour.The blocking buffer was discarded, and Anti-phospho-Axl (Y702) (D12B2) rabbit monoclonal antibody (Cell Signaling Technology, Inc., catalog No. 5724) was reacted at a concentration of 1/1000 with the plate, which was then left standing overnight at 4° C. Each well was repetitively washed with a wash buffer five times, and Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Inc., catalog No. 711-035-152) was reacted at a concentration of 1/2000 with the plate at room temperature for 1 hour. A similar washing operation was carried out, and Super Signal ELISA pico chemiluminescent substrate (Thermo Fisher Scientific, Inc., catalog No. 37069) was added at 0.05 ml/well to the plate and gently stirred, followed by incubation for 20 minutes. Then, developed light was measured using ARVO sx (PerkinElmer Inc.) to measure the pAxl (Y702) level. |
| Affinity data for this assay | |
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