| Assay Method Information | |
| | Standard LATS2 HTRF Assay |
| Description: | Human LATS2 catalytic domain which contains the amino acids G553-V1088 (accession NP_055387) was purified in-house. The LATS2 catalytic domain was co-purified with Mob1b (accession NP_775739). The LATS2 biochemical HTRF assay was performed using the HTRF KinEASE-STK S1 Kit (Cisbio, Cat #62ST1PEC), following the protocol from the manufacturer. Compounds were dispensed by the Echo Liquid Handler (Labcyte) into a white 384-well plate (PerkinElmer, Cat #6008289). 3 uL of 2×LATS2 enzyme solution was added to the compounds, following by a 10 minute incubation at room temperature. Then, 2×ATP and STK S1 peptide solution was added to initiate the one hour enzyme reaction at room temperature. The final condition of the reaction was 0.2 nM LATS2, 50 μM ATP, 0.5 μM STK S1 peptide in 50 mM HEPES pH7.2, 10 mM MgCl2, 0.1% BGG, 0.005% Brij-35, 1 mM DTT. The reaction was quenched by adding 6 uL of the detection mixture which contained Streptavidin XL665 and STK Antibody-Cryptate (Cisbio), incubate for 1 hour at room temperature. The HTRF (665 nm/620 nm) signal was read on the Envision plater reader (PerkinElmer). The IC50 values were determined by fitting the % inhibition with a nonlinear four-parameter logistical equation. The Ki values were calculated using the Cheng-Prusoff equation for a competitive inhibitor, IC50═Ki (1+S/Km)+M [E] with ATP Km for LATS2=105 μM. |
| Affinity data for this assay | |
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