| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | Assay Method: (1) A compound stock solution was prepared and diluted 3× to give a compound dilution; 10 nL of the compound dilution was transferred to a 384-well plate (784075, Greiner) by Echo 550;(2) The plate was sealed, and centrifuged at 1,000 g for 1 min;(3) 2×EGFRL858R/T790M/C797S protein working solutions were prepared with 1× kinase buffer, respectively;(4) 5 μl of the 2×EGFR protein working solution was added to the 384-well plate from step (2), centrifuged at 1,000 g for 30 s, and allowed to stand at room temperature (mixed thoroughly) for 10 min;(5) A mixture of 2× TK-substrate-biotin (2 μM) and ATP was prepared with 1× Kinase buffer;(6) 5 μL of the TK-substrate-biotin and ATP (the mixture prepared in step (5)) was added to the 384-well plate from step (4) to initiate the reaction;(7) The mixture was centrifuged at 1,000 g for 30 s; the plate was sealed, and allowed to stand (and reacted) at room temperature for 40 min;(8) 4×Sa-XL 665 and TK-antibody-Cryptate were prepared with detection buffer;(9) 5 μL of the Sa-XL 665 and 5 μL of the TK-antibody-Cryptate were added successively to the 384-well plate from step (7);(10) The mixture was centrifuged at 1,000 g for 30 s, and allowed to stand (react) at room temperature for 1 h;(11) Fluorescence values were read at 615 nm and 665 nm by enzyme labeling instrument (PerkinElmer, 74785). |
| Affinity data for this assay | |
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