| Assay Method Information | |
| | Kinase Activity Assay |
| Description: | 1.3 Preparation of 1× Kinase Buffer4 volume of distilled water was added to 1 volume of enzyme buffer 5×; 5 mM MgCl2; 1 mM DTT.1.4 Screening Methoda) 1 μL 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) 2× Ret wt (0.04 ng/μL), 2× Ret V804M (0.2 ng/μL) and 2×RET G810R (2 ng/μL) in 1× kinase buffer were prepared;e) 5 μL of 2× Ret wt, Ret V804M or RET G810R was added to a 384-well test plate;f) 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 a solution of 5×ATP (50 μM) in kinase buffer were prepared with 1× kinase buffer;h) the reaction was initiated by adding 2 μL of STK-substrate-biotin and 2 μL 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 detection buffer was prepared;k) 5 μL 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) the plate was read for the fluorescence signal values of 620 nm (Cryptate) and 665 nm (XL665) on Envision 2104 plate reader or BioTek microplate reader.1.5 Data Analysis |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |