| Assay Method Information | |
| | Radiometric Kinase Assay |
| Description: | All kinase assays were performed as previously described5 at −4° C. in a final volume of 18 μL, containing 2 μL of compounds according to the invention diluted in Tris-HCl-glycerol, 0.05% Tween, 3 μL of CK2a (36 ng) and a mixture of 1 mM peptide substrate, 10 mM MgCl2 and 1 μCi [γ32P]-ATP. Final concentration of ATP was 10 μM if not stated otherwise. Assays were performed under linear kinetic conditions for 5 min at room temperature before termination by the addition of 60 μL of 4% TCA. 32P incorporation in peptide substrate was determined by spotting the supernatant onto phospho-cellulose paper disks (Whatman P81,4 cm2). The disks were washed three times in cold 0.5% phosphoric acid, 5 minutes on a rocking platform per wash then dried and their radioactivity was measured. Percentage inhibition was calculated relative to a DMSO control and IC50 or Ki values were calculated using test GraphPad Prism 8.The peptide substrates employed for different assays were a canonical CK2 peptide substrate Seq. ID No. 1: RRREDEESDDEE, phosphorylated equally by CK2a subunit and CK2 holoenzyme (CK2(3-independent peptide substrate), and Seq. ID No. 2: MSGDEMIFDPTMSKKKKKKKKP exclusively phosphorylated by CK2 holoenzyme (CK2(3-dependent peptide substrate).6GST-SIX1 phosphorylation assay was performed following the CK2 radiometric kinase assay. CK2α (200 nM) was incubated with increasing concentrations of CK20 in the absence or presence of AB668 followed by the addition of GST-SIX1 (3.7 μg), 10 mM MgCl2 and 1 μCi [γ32P]-ATP. Final concentration of ATP was 100 μM. Samples were analyzed by SDS PAGE and subjected to autoradiography. Phosphoproteins were quantified by densitometry scanning. |
| Affinity data for this assay | |
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