| Assay Method Information | |
| | LATS2 Biochemical Caliper Assay |
| Description: | Human LATS2 kinase domain protein was purchased from Carnabio (catalogue number 01-124), which harbors the catalytic domain of amino acids 553-1088, and was co-purified with human His-tagged MOBKL1A (NP_775-739). 5 microlitre of enzyme buffer, 100 nL of compounds and 5 microlitre of localising aid buffer were added into 384-well plates. The final assay reaction mix contains 100 mM HEPES, pH7.5, 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl2, 10 micromolar Sodium Orthovanadate, 10 micromolar Beta-Glycerophosphate, 400 micromolar ATP, 1% DMSO, 1.1 nM LATS2 enzyme (Carnabio, 01-124), 1 micromolar localising aid of FAM-KKLRRTLSVA-COOH (SEQ ID NO: 24) (NanoSyn). The plates were incubated at 25° C. for 3 hours. 40 microlitre of stop buffer with 25 mM EDTA (NanoSyn) was added to each well to terminate the reaction. Localising aids and products were separated electrophoretically using the microfluidic-based Caliper Labchip 3000 Drug Discovery System (Caliper Life Sciences). Plates were read using blue laser excitation and green fluorescence detection and quantified by fluorescence intensity. The IC50 is measured when the effect of the compound reduces the product fluorescence signal by 50%. |
| Affinity data for this assay | |
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