| Assay Method Information | |
| | In Vitro Kinase Assay |
| Description: | In vitro kinase assays were performed as described below:Recombinant His-tagged ULK1/Atg13 kinase complex was expressed in SH9 insect cells, and affinity-purified by Ni-NTA beads, aliquoted and stored at −80° C.Compounds were dissolved in DMSO to make 10 mM stock solution, aliquoted and stored at −80° C.1× kinase buffer was prepared: 20 mM Tris-HCl, pH7.5, 100 mM NaCl, 5 mM MgCl2, 0.5 mg/ml BSA, 5 mM DTT, 0.1% Tween-20.For compound screening experiment, 1 μl of the 10 mM compound stock solution was added using P2.5 pipette into 99 μl 1×kinase buffer to make 100 μM working solution.For compound titration experiment, 100 μM compound solution was further diluted (with 1×kinase buffer containing 1% DMSO) in a series manner.30 mM MBP (Myelin basic protein) 1-20 peptide (sequence: ASQKRPSQRSKYLATASTMD; SEQ ID NO: 1) was prepared in 1× kinase buffer.500 μM ATP was prepared in 1× kinase buffer. Pre-incubation reactions were set up as shown in the Table below and pre-incubated for 10 min at room temperature.Pos Neg Test1x kinase buffer 4 μl 8 μl 4 μl20 nM ULK1 4 μl 0 μl 4 μl30 mM MBP 4 μl 4 μl 4 μlCompound or 4 μl 1% DMSO 4 μl 1% DMSO 4 μl compound DMSO control TOTAL 16 μl 16 μl 16 μl4 μl of 500 μM ATP was added to the pre-incubation reaction (thus the final reaction volume is 20 μl) and incubated for 75 min at room temperature.20 μl Kinase-Glo Plus (Promega) was added to the reaction (warmed to RT), mixed well and incubated at room temperature for 15 min.20 μl reaction was pipetted into each well of a white 384-well plate.the 384-well plate was subjected to brief centrifugation, and luminescence was read in a plate reader. |
| Affinity data for this assay | |
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