| Assay Method Information | |
| | MNK Biochemical Enzymatic Assay |
| Description: | This protocol establishes the binding assays for MNK1 and MNK2 using ADP-Glo assay. MNK phosphorylates the substrate and converts ATP to ADP, which was detected by Envision and used to reflect the reminding activity of MNK. Reagents and equipment used in the assay are listed below, followed by the protocol.Number Name Vendor Cat# 1 HEPES Life Technologies 15630-080 2 NaCl Sigma S5886-1Kg 3 MgCl2 Sigma M1028 4 BSA Sigma B2064-50G 5 Tween-20 Bio-RAD 170-6531 6 ADP-Glo Kinase Assay Promega V9101 7 MNK1 Carna 8 MNK2 Carna 9 ATP Promega V915B10 Substrate peptide NJ peptide 11 Topseal A PerkinElmer E534112 OptiPlate-384 PerkinElmer 600729013 Envision Perkin Elmer 210414 Centrifuge Eppendorf 5810R15 Echo 550 Liquid Handler Labcyte Echo 550a) Add 50 μL compound to 384-well dilution plateb) Dilute compound 1:3 in succession in DMSO for each column for 10+0 pts (refer to dilution plate map)c) Transfer 0.1 μL diluted compound solution in each row to 384 assay plate using Echo, each column containing 2 replicates (refer to assay plate map)d) Add 5 μL enzyme working solution to 384-well assay plate, centrifuge 1000 RPM for 1 mine) Incubate at 25 C. for 15 minf) Add 5 μL substrate working solution to initiate reactiong) Incubate at 25 C. for 60 minh) Add 10 μL ADP Glo reagent, centrifuge 1000 RPM for 1 mini) Incubate at 25 C. for 60 minj) Add 20 μL kinase detection reagent, centrifuge 1000 RPM for 1 mink) Incubate at 25 C. for 60 minl) Read on Envision for US LUM as RLUm) Data analysis: IC50s were determined based on a non-linear regression analysis of data collected. |
| Affinity data for this assay | |
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