Labchip ez reader

Kinase activities were quantified with Microfluidic mobility shift assay (MMSA) using 1 mM ATP and 1 mM LRRKtide (RLGRDKYKTLRQIRQ-amide, GeneCust) as substrates. Two stock solutions were prepared, each containing 2× concentration: substrate solution (1900 µM LRRKtide, 100 µM Fluorescein-LRRKtide, 2 mM ATP) and enzyme solution (100 nM LRRK2 [functional protein concentration], 20 mM MgCl2, 1 mM GDP). Both stock solutions were prepared using standard kinase buffer (25 mM Tris–HCl, pH 7.5, 50 mM NaCl, 0.1 mg/ml BSA, 1 mM DTT). Reactions were started by mixing both solutions in a 1 : 1 ratio in a 384 well-plate and measurements were performed at 30°C. Substrate conversions were monitored for 60–90 min using a LabChip EZ Reader (PerkinElmer). The slope (conversion rate, [m] = %/min) of the percental conversion plotted against the time was determined using a linear fit model of GraphPad Prism 6 and was converted into reaction velocity ([v0] = µmol/min) and further into observed turnover numbers k_obs [min⁻¹]. Experiments were performed at least in duplicates of duplicates for two independent LRRK2 expressions. To determine significant differences between LRRK2 wt and mutant activity a two-sided unpaired t-test (parametric) with a confidence interval of 95% was performed.

Inhibition of KAT3B acetyltransferase activity by ngKATIs was assessed by a separation-based assay^{70 (link)}. Reactions consisted of buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM EDTA, 2 mM DTT, 0.05% Triton-X-100) with KAT3B (P300, 150 nM) and FITC-Ahx-RGKGGKGLGKGG [Ahx = 6-aminohexanoic acid] substrate (2 μM) were plated in 384-well microplates and equilibrated at RT for 10 min in the presence or absence of inhibitor. Reactions were initiated by addition of acetyl-CoA (1 μM final concentration) with 30 μL final assay volume and quenched during steady-state kinetics (<15% product accumulation) by addition of 5 μL of 0.5 M neutral hydroxylamine. Quenched reaction aliquots were then transferred to a PerkinElmer Lab-Chip EZ-Reader instrument for microfluidic electrophoresis and fluorometric analysis. Optimized separation conditions were downstream voltage −500 V, upstream voltage −2500 V, and pressure −1.5 psi. Percent conversion is calculated by ratiometric measurement of substrate/product peak heights, corrected for nonenzymatic background acetylation. Percent activity KAT3B represents acetylation relative to vehicle control. Concentration responses were analyzed by nonlinear least-squares regression fits to a 4PL equation. Data are mean ± SD from three technical replicates.

AXL, MER, MET, and TRKA kinase inhibition by DS-1205b was determined using the LabChip EZ Reader (PerkinElmer Inc., Waltham, MA, USA) in the presence of 1 mM ATP. DS-1205b dimethyl sulfoxide (DMSO) solution was added to kinase solution and the mixture was incubated at room temperature for 20 min. After the addition of ATP, the reaction plates were incubated at 28°C for 45 min. The kinase panel was evaluated by mobility shift assay (Carna Biosciences, Inc., Natick, MA, USA). The IC₅₀ was determined.

The GSK-3β inhibition assay was performed by calliper mobility shift assay using the method described previously⁶⁴ (link)^,75 and AR-A014418 was used as a positive control. In brief, compounds or AR-A014418 were tested from 1 μM or 5 μM, 3-fold dilution for IC₅₀ determination. GSK-3β protein and the tested compound were loaded in 384-well plate (Corning). After incubation for 10 min, the FAM-labeled peptide 15 (GL Biochem, Shanghai, China) and ATP prepared in the reaction buffer were added and ran for 1 h at 28 °C. Stop buffer (25 μL) was added and conversion data were collected on a LabChip EZ Reader (PerkinElmer, Shanghai, China) at each concentration through the direct detection of both substrate and product via Laser-Induced Fluorescence (LIF) at 492 nm. The IC₅₀ values were calculated from dose-response curves using XLfit (curve fitting software for Excel).

Full-length recombinant PTPN2 or PTPN1 were incubated with increasing concentrations of AC484 or A-650 for 10 min at room temperature. Oregon green-labelled insulin receptor probe (sequence: (OG488)-DPEG2-T-R-D-I-(PY)-E-T-D-Y-Y-R-K-K-CONH2) was added and incubated for another 10 min. Finally, the reaction was quenched with water and a potent known PTPN2/PTPN1 inhibitor. Fluorescent signals of product and probe were subsequently measured on a LabChip EZ Reader (Perkin-Elmer).

Human Janus kinase (JAK) activity was determined using a microfluidic assay to monitor phosphorylation of a synthetic peptide by the recombinant human kinase domain of each of the four members of the JAK family, JAK1, JAK2, JAK3, and TYK2. Reaction mixtures contained 1 μM of a fluorescently labeled synthetic peptide and 1 mM ATP. Each assay condition was optimized for enzyme concentration and room temperature incubation time to obtain a conversion rate of 20 − 30% phosphorylated peptide product. Reactions were terminated by the addition of stop buffer containing EDTA. Utilizing mobility shift technology (PerkinElmer LabChip EZ Reader), each assay reaction was sampled to determine the level of phosphorylation. This technology is separation-based, allowing direct detection of fluorescently labeled substrates and products with separations controlled by a combination of vacuum pressure and electric field strength optimized for the peptide substrate. See the Supporting Information for commercial and sequence information^{13 (link),21} .