Biomol green assay

Initial rates of WIP1 phosphatase activity were determined by incubating WIP1(1–420) (26 nm) with various concentrations of [h-ATM (1976–1986-GY, 1981pS)] peptide 6 in the buffer: 50 mm Tris-HCl (pH = 7.5), 30 mm MgCl₂, 0.1 mm EGTA, 0.1 mg/ml BSA, 1 mm CHAPS and 1 mm DTT at 30 °C for 7 min. The phosphate reaction product was measured using the BIOMOL green assay (Enzo Lifesciences), following the manufacturer's protocol. To estimate K_m and k_cat values, the initial rates were fit to the Michaelis-Menten equation in GraphPad Prism 7.01. Inhibition of phosphatase activity of WIP1(1–420) (26.0 nm) were performed as described previously (30 (link)). Inhibition of phosphatase activity was determined in the above buffer and 1% DMSO using 90.0 μm peptide 6.

GTPase activities of FtsZ-YFP-mts were determined by measuring released inorganic phosphate using BIOMOL^® GREEN assay (Enzo Life Sciences USA). GTP hydrolysis reaction was performed in polymerization buffer (50 mM Tris/HCl, 300 mM KCl, 5 mM Mg²⁺, pH7.5) using FtsZ-YFP-mts at 5 μM with 1 mM GTP. Reactions were performed every 20 s for a total time of 120 s. After 30 min of incubation with BIOMOL^® GREEN at room temperature, the absorbance of the samples at 620 nm was monitored by TECAN plate reader. The phosphate release concentrations were calculated based on a phosphate standard curve.

Recombinant human PHOSPHO1 (50 ng) was generated as previously described [27 (link)] and incubated with varying concentrations of the aforementioned PPIs and H2RAs in experimental assay buffer (20 mM Tris, 2 mM MgCl₂ & 25 µg/ml BSA) at 37 °C for 15 min. Using the BIOMOL® Green assay (Enzo, Exeter, UK), standards (0-2 nM) and samples were then incubated with 2.5 mM β-glycerol phosphate for 30 min at 37 °C with gentle agitation [27 (link)]. The reaction was stopped using 100 µl BIOMOL® Green and after being left for 30 min at room temperature, the absorbance was read using spectrophotometry at 630 nm. For TNAP, 2 ng recombinant human TNAP (R&D Systems, Abington, UK), was incubated with varying concentrations of the aforementioned PPIs and H2RAs in experimental assay buffer (1 M diethylamine hydrochloride, 1 mM MgCl₂ and 20 µM ZnCl₂). Using the BIOMOL® Green assay, standards (0-2 nM) and samples were then incubated with 0.5 mM p-nitrophenyl phosphate (pNPP) for 30 min at 37 °C with gentle agitation. The reaction was stopped using 100 µl BIOMOL® Green and after being left for 30 min at room temperature, the absorbance was read using spectrophotometry at 630 nm.