| Assay Method Information | |
| | pEGFR assay |
| Description: | This assay quantifies the phosphorylation of EGFR at Tyr1068 and was used to measure the inhibitory effect of compounds on the transgenic EGFR del19 T790M C797S protein in Ba/F3 cells. Murine Ba/F3 cells were grown in RPMI-1640 (ATCC 30-2001) + 10 % FCS + 10 ng/mL IL-3 at 37 蚓 in 5 % CO2 atmosphere and transduced with a retroviral vector encoding EGFR del19 T790M C797S. Transduced cells were selected using puromycin. Following selection, IL-3 was withdrawn and IL-3 independent cells cultured. p-EGFR Tyr1068 was determined using the AlphaScreen Surefire pEGF Receptor (Tyr1068) Assay (PerkinElmer, TGRERS). For the assay, Ba/F3 EGFR del19 T790M C797S cells were seeded in DMEM medium with 10 % FCS.60 nL compound dilutions were added to each well of Greiner TC 384 plates using the Echo platform. Subsequently, 60.000 cells/well in 60 無 were added. Cells were incubated with compound for 4 h at 37 蚓. Following centrifugation and removal of the medium supernatant, 20 無 of 1.6-fold lysis buffer from TGR/Perkin Elmer kit with protease inhibitors was added. The mixture was incubated at room temperature with shaking (700 rpm) for 20 min. After centrifugation, 4 無 of the lysate were transferred to Proxiplates. 5 無 of Acceptor Mix (Activation Buffer diluted 1:25 in combined Reaction Buffer 1 and Reaction Buffer 2 (TGRERS Assay Kit, PerkinElmer) plus 1:50 of Protein A Acceptor Beads 6760137) were added to each well. Plates were shaken for 1 min (1400 rpm) and incubated for 2 h at room temperature in the dark.3 無 of donor mix (AlphaScreen Streptavidin-coated Donor Beads (6760002, PerkinElmer) 1:50 diluted in Dilution Buffer (TGRERS Assay Kit, PerkinElmer) were added to each well. Plates were shaken for 1 min (1400 rpm) and incubated for 2 h at room temperature in the dark. Plates were subsequently analyzed using an Envision reader platform. Results were computed in the following way: The ratio of the value of the test compound and the value of the negative control (DMSO) was calculated. IC50 values are computed from these values in the MEGASTAR IC50 application using a 4 parametric logistic model. |
| Affinity data for this assay | |
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