| Assay Method Information | |
| | Determination of the Inhibitory Activity of the Compound of the Present Disclosure on RET Enzyme |
| Description: | (1) 1× enzyme reaction buffer: 5× enzyme reaction buffer was diluted with ultrapure water (UPW) at 4:1, and then 25 nM SEB was added.(2) 5×RET kinase: RET kinase was diluted with 1× enzyme reaction buffer to 2.5 nM.(3) 5×ATP: ATP was diluted with 1× enzyme reaction buffer to 50 μM.(4) 5× substrate: TK substrate-biotin was diluted with 1× enzyme reaction buffer to 2,500 nM.(5) 2.5× compound: The highest final concentration was 2,500 nm, 3-fold gradient dilution was conducted to obtain 10 dilution points, and a final DMSO concentration was 0.4%.(6) 4× Steptavidin-XL665: Steptavidin-XL665 was diluted with 1× enzyme reaction buffer to 250 μM.3. Experimental Process(1) 5×RET kinase was added to a 384-well plate at 2 μl/well (final concentration of 0.5 nM), and a blank group (enzyme reaction buffer) was set as a negative control.(2) A 2.5× compound solution was added at 4 μl/well, and then the plate was incubated at room temperature for 30 min. A DMSO group was set as a positive control.(3) 5× substrate (at 2 μl/well, final concentration: 500 nM) and 5×ATP (at 2 μl/well, final concentration: 10 μM) were added to start a reaction, and the reaction was conducted at room temperature for 30 min.(4) TK Antibody-Cryptate (at 5 μl/well) and 4× Steptavidin-XL665 (at 5 μl/well, final concentration: 62.5 μM) were added, and the plate was incubated at room temperature for 60 min.(5) An HTRF signal was read on Envision.4. Data Analysis |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |