| Assay Method Information | |
| | MASTL Activity Assay |
| Description: | Wild-type human MASTL (0.5 nM) was incubated in assay buffer (30 mM HEPES, 100 mM NaCl, 0.5 mM EGTA, 10 mM MgCl2, 0.01% Tween-20, 0.5 mM TCEP, pH 7.5) with biotin tagged 40-mer ENSA peptide (10 nM), test compound and ATP (18 μM) for 15 minutes at room temperature. Test compounds were assayed using a 10-point dose range consisting of a 0, DMSO control and 9 sequential doses consisting from 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μM. The DMSO concentration was the same (0.1%) in all samples. An equal volume of detection buffer (assay buffer+0.53 nM Ab-K, 2.5 nM SA-D2, 20 mM EDTA and 100 mM KF) was added to stop the reaction to make a final volume of 20 μl. Activity was measured using a plate reader (BMG Labtech Pherastar) by the FRET signal generated between SA-D2 (streptavidin-D2) and Ab-K (anti-phospho-Serine 67 ENSA antibody (rabbit polyclonal, using standard techniques by a commercial supplier) conjugated to Cryptate). HTRF reagents (CisBio) were prepared as per the manufacturer recommendations. The 40-mer biotin-tagged ENSA peptide (synthesised by Peptide Protein Research) used was based around Serine 67 on ENSA (YPSLGQKPGGSDFLMKRLQKGQKYFDSGDYNMAKAKMKNK). |
| Affinity data for this assay | |
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