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| | LATS1 Biochemical Caliper Assay |
| Description: | The LATS1 biochemical Caliper assay was performed as following.Human LATS1 kinase domain protein was purchased from Carnabio (catalogue number 01-123; lot 15CBS-0098D). Human LATS1, catalytic domain [589-1130(end) amino acids of accession number NP_004681.1] was co-expressed as N-terminal GST-fusion protein (90 kDa) with human His-tagged MOBKL1A [1-216(end) amino acids of accession number NP_775739.1] using baculovirus expression system. GST-LATS1 was purified by using glutathione sepharose chromatography. The substrate (Fluo-SGKtide; Peptide for LATS1; lot BS-41067) has the following sequence: 5-Fluo-Nva-KKRNRRLSVA-amide (SEQ ID NO: 27) x TFA and was purchased from Biosyntan.The reaction is performed in reaction buffer containing 50 mM Hepes pH 7,5; 0.02% Tween20; 0.02% BSA; 1 mM DTT; 10 uM Na3VO4 and 10 mM beta-Glycerolphosphat and fresh added 1 mM MgCl2 and qsp H2O.The substrate solution (2× conc.) in Reaction Buffer contains 300 μM ATP and 4 μM Fluo-SGKtide.The kinase solution (2× conc.) in Reaction Buffer contains 20 nM LATS1 kinase. 4.5 μL of 2× conc. Kinase solution, 50 nL of 1.8 mM compounds and 4.5 μL of substrate solution were added into 384-well plates black small volume from Greiner and incubated at 32° C. for 1 hour. 15 μL of stop buffer containing 100 mM Hepes pH 7,5; 5% DMSO; 0.1% Coating reagent; 10 mM EDTA and 0.02% Brij35 and qsp H2O to each well to terminate the reaction.Substrates and products were electrophoretically separated using the microfluidic-based Caliper EZ Reader System (Caliper Life Sciences) using a 12 sipper chip (cat 760404). The separation takes place in Coating Buffer (idem Stop buffer) and containing 0.1% Coating reagent CR3 and 0.5% coating reagent CR8 (Perkin Elmer).Plates were read using a LED with an excitation at 488 nm and a detection at 520 nm to 5 quantify the 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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