Malachite green assay

Phosphate released from synthetic phosphopeptide substrates by SHP2 was measured using Malachite Green assay (MAK307, Sigma–Aldrich). The dephosphorylation reaction contained 50 nM wild-type SHP2(1–525), 6 µM bisphosphorylated IRS1 ligand for activating SHP2, and varying concentrations of phosphopeptide substrates (0–0.5 mM) in a final volume of 40 µl buffer (60 mM HEPES [pH 7.2], 75 mM KCl, 75 mM NaCl, 1 mM EDTA, 0.05% Tween 20, and 2 mM DTT). The reactions were incubated at room temperature for 5 min in a 384-well plate in triplicate and terminated by the addition of 10 µl malachite green solution (Sigma–Aldrich, Catalog # MAK307) to each well. After further incubation for 30 min at room temperature for color development, absorbance was measured at 620 nm on a plate reader. The amount of phosphate released was determined by comparison with a malachite green standard curve. The reaction rate, defined as micromoles of phosphate released per minute, reflects the dephosphorylation activity of SHP2 toward its phosphopeptide substrates. The sequences of the peptides used in the assay were:

A malachite green assay (Sigma-Aldrich, St. Louis, MO, USA) was used to measure the ATPase activity of CDSecA2. Three independent experiments of triplicate wells of a 96-well plate were set up containing either 2, 4 or 6 µL of 0.75 mg/mL CDSecA2 in 30 mM Tris and 400 mM NaCl pH 7.5, and the samples were brought to a final volume of 10 µL with assay buffer (40 mM Tris, 80 mM NaCl, 8 mM Mg(CH₃COO)₂ and 1 mM EDTA, pH 7.5). Reaction mixes composed of 20 µL of assay buffer and 10 µL of 4 mM ATP were added to each sample, background blank and negative control well. The reactions were incubated for 30 min. Formation of inorganic phosphate was monitored spectrophotometrically by the increase in absorbance at 620 nm at 30 min after adding 200 µL of malachite green reagent. The inorganic phosphate concentrations generated in the reaction mixtures were calculated using a standard curve. The assay was conducted at 25 °C, and the average rate of ATP hydrolysis and the standard error were determined.