Assay Method Information
Assay Name:  Inhibitory Activity Assay
Description:  1. Experimental Purpose:The inhibitory activities of the series of compounds against Ret wt, VEGFR2, CCDC6-RET, Ret M918T, Ret V804L, and Ret V804M were tested by HTRF method, and IC50 values were determined.2, The assay agents and consumables used are as shown below:1) HTRF KinEASE-TK kit (Cisbio, 62TK0PEC)2) Ret wt (Invitrogen, PV3082)3) VEGFR2 (invitrogeon, PV3660)4) CCDC6-RET (Signalchem, R02-19BG-10)5) RetM918T (Signalchem, R02-12JG-10)6) Ret V804L (Signalchem, R02-12BG-10)7) Ret V804M (Signalchem, R02-12GG-10)8) MgCl2 (Sigma, M1028)9) ATP (Promega, V910B)10) DTT (Invitrogen, P2325)11) DMSO (Sigma, D8418)12) 384-well plate, white, low volume, round-bottom (Greiner, 784075)13) 384-Well Polypropylene microplate, Clear, Flatt Bottom, Bar Code (Labcyte, P-05525-BC)14) 96-well polypropylene plate (Nunc, 249944)15) Plate shaker (Thermo, 4625-1 CECN/THZ Q)16) Centrifuge (Eppendorf, 5810R)17) Envision 2104 multi-label Reader (PerkinElmer, 2104 Oct. 1)18) Echo (Labcyte, 550)3. Experimental procedure3.1 Preparation of 1× Kinase Reaction Buffer:1 volume of 5× kinase reaction buffer and 4 volumes of water; 5 mM MgCl2; 1 mM DTT;1 mM MnCl2.3.2 10 nl of diluted compound was transferred to per well in the Echo 550 reaction plate (784075, Greiner);3.3 The reaction plate was sealed with a sealing film and centrifuged at 1000 g for 1 minute.3.4 1× Enzyme reaction buffer was used to prepare 2× Kinase.3.5 5 μl of kinase was added to each well in the reaction plate (prepared in step 3). The plate was sealed with a sealing film, centrifuged at 1000 g for 30 seconds, and then left at room temperature for 10 minutes.3.6 4×TK-substrate-biotin and 4×ATP were prepared with 1× enzyme reaction buffer, and then mixed uniformly. To the reaction plate was added 5 μl of the mixture of K-substrate-biotin/ATP.3.7 The plate was sealed with a sealing film, centrifuged at 1000 g for 30 seconds, and then left for reaction at room temperature for 40 minutes.3.8 4× Sa-XL 665 (250 nM) was prepared using HTRF assay buffer.3.9 5 μl of Sa-XL 665 and 5 μl of TK-antibody-Cryptate were added to each well, centrifuged at 1000 g for 30 seconds, and reacted at room temperature for 1 hour.3.10 Fluorescence signals at 615 nm (Cryptate) and 665 nm (XL665) were read with Envision 2104.4. Data Analysis4.1 Calculation of the ratio of each well (Ratio_665/615 nm)4.2 The inhibition rate was calculated as follows:The⁢⁢inhibition⁢⁢rate⁢⁢of⁢⁢the⁢⁢compound⁢⁢(%⁢⁢inhibition)= [1-Ratiocompound-Ratio_positive⁢⁢controlRatio_negative⁢⁢control-Ratio_positive⁢⁢control]=100
Affinity data for this assay
 
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