| Assay Method Information | |
| | HTRF Assay |
| Description: | a) 10 nL of compound dilution was transferred to each well of the test plate;b) the compound plate was centrifuged at 1000 g for 1 min;c) the test plate was sealed;d) 5× Ret-wt (0.2 ng/L), 5× Ret V804M (0.5 ng/L), and 2.5× RET G810R (2.5 ng/L) in 1× kinase buffer were prepared;e) 2 μL of 5× Ret-wt, 2 L of Ret V804L, or 2 L of RET G810R was added to a 384-well test plate;f) 4 μL of 1× kinase buffer was added to each well of the test plate, and the sample plate was centrifuged at 1000 g for 30 s and left to stand at room temperature for 10 min;g) a solution of 5× TK-substrate-biotin (5 μM) in kinase buffer and 5× ATP (50 μM) in kinase buffer were prepared;h) the reaction was initiated by adding 2 L of STK-substrate-biotin and 2 L of ATP (prepared in step g);i) the sample plate was centrifuged at 1000 g for 30 s, and the test plate was sealed and left to stand at room temperature for 30 min;j) 4× Sa-XL 665 (250 nM) in HTRF assay buffer was prepared;k) 5 L of Sa-XL 665 and 5 μL of TK-antibody-Cryptate (prepared in step i) were added to each well of the test plate;l) the plate was centrifuged at 1000 g for 30 s, and left to stand at room temperature for 1 h; andm) fluorescence signal values of 615 nm/620 nm (G810R) (Cryptate) and 665 nm (XL665) were read on an Envision 2104 microplate reader or BioTek microplate reader. |
| Affinity data for this assay | |
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