| Assay Method Information | |
| | Inhibitory Activity of Compounds on TRKA, TRKB and TRKC |
| Description: | 2.1 Formulation of 1× Kinase Reaction Buffer1 volume of 5× kinase reaction buffer and 4 volumes of water were used together with 5 mM MgCl2; and 1 mM DTT.2.2 Preparation of Kinase and SubstrateFormulation of 2.5× Substrate MixtureATP concentration Poly (4:1 Glu, Tyr)Kinase [μM] Peptide[μM]TRKA 5 0.03TRKB 5 0.03TRKC 5 0.032.3 Test Procedure on Inhibitory Activity of Compounds1) Dilute the compound by 4 times with DMSO in a dilution plate, in which the initial concentration of the compound was 1000 nM.2) Dilute the compound by 50 times with a 1× kinase reaction buffer and incubate it on a shaker for 20 minutes.3) Formulate 2×TRKA with 1× kinase reaction buffer.4) Add 2 μl of TRKA kinase (prepared in step 3) to each well of a reaction plate.5) Add 1 μl of the compound diluted in buffer to each well, seal the plate with a sealing film, centrifuge it at 1000 g for 30 seconds, and incubate it at room temperature for 10 minutes.6) Formulate a 4×ATP & sub mixture with 1× kinase reaction buffer, and add 1 μl of the 4×ATP & sub mixture to the reaction plate.7) Seal the plate with a sealing film, centrifuge it at 1000 g for 30 seconds, and incubate it at room temperature for 60 minutes.8) Transfer 4 μL of ADP-Glo to the 384-well reaction plate, centrifuge it at 1000 rpm/min for 1 minute, and incubate it at 25° C. for 40 minutes.9) Transfer 8 μL of Detection solution to the 384-well reaction plate, centrifuge it at 1000 rpm/min for 1 min, and incubate it at 25° C. for 40 min.10) Use Biotek multi-function plate reader to read RLU (Relative luminescence unit) signal. The signal intensity is used to characterize the degree of kinase activity. |
| Affinity data for this assay | |
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