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Assay Method Information |
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| | Enzyme Assay |
| Description: | In this assay, CSNK1D phosphorylates a substrate peptide PLSRTL-pS-VASLPGL in the presence of ATP. This substrate peptide has been modeled after the sequences surrounding three main cyclic AMP-dependent protein kinase sites of glycogen synthase. This assay monitors CSNK1D kinase activity by measuring the amount of ADP produced in the assay. A substrate mix is prepared by diluting peptide substrate (final concentration 150 μM) with ATP (final concentration 20 μM) in assay buffer (50 mM Tris/HCl pH 7.4+10 mM MgCl2+1 mM DTT+0.1% BSA). The substrate mix is added to each well of a low volume, 384-well, white opaque plate. Test compounds were diluted in HBSS and added in a dose-response to the plate. To start the reaction, 2 nM of constitutively active human recombinant GST cleaved CSNK1D (University of Dundee, clone DU 19064, stored at 0.28 mg/mL in 50 mM Tris/HCl pH 7.5, 150 mM NaCl, 270 mM Sucrose, 0.1 mM EGTA, 0.1% 2-mercaptoethanol, 0.02% Brij-35.1 mM benzamidine, 0.2 mM PMSF) was added to each well and the plate centrifuged for 5 minutes at 1500 rpm. The total volume of each reaction is 5 ul (2 μL of substrate mix, 1 μL of diluted compounds, and 2 μL of human recombinant CSNK1D). The plates are incubated for 45 minutes at room temperature. |
| Affinity data for this assay | |
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