Measure it thiol assay kit

To measure free thiol groups in cells untreated or CAP-treated for 1 minute, the cells were incubated with maleimide coupled to the fluorescent dye Alexa 633 (Molecular Probes, Thermo-Fisher Scientific). Thiol binding was measured by flow cytometry (Becton Dickinson, Durham NC, USA) according to a previously described procedure [20 (link)]. The thiol content in the lysed proteins was measured using a fluorescent probe in the Measure-IT^™ Thiol Assay Kit (Molecular Probes, Thermo-Fisher Scientific). The fluorescence was recorded in a fluorometer at excitation/emission wavelengths of 494/517 nm (Varioskan Flash; Thermo-Fisher Scientific) [44 (link)]. The thiol content in each sample was calculated from a standard curve and normalized to the total protein, which was quantified by bicinchoninic acid assay protein assay (#23227, Thermo-Fisher Scientific).

To measure production of H₂O₂, Hpx⁻ strains ΔkatGΔkatEΔahpCF E. coli and ΔkatAΔahpCF B. subtilis were used, which allow accumulation and measurement of H₂O₂[12] (link), [15] (link)–[17] (link). Bacteria (50 µl) were incubated as for gene expression arrays with albumin (control), PGRP, paraquat, or diamide (at concentrations given in Results), for 15–120 min (15 min was the optimum time for the highest induction of H₂O₂, determined in preliminary experiments), and total amount of H₂O₂ was determined using fluorescent Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (InVitrogen/Molecular Probes) according to the manufacturer's instructions. To measure depletion of thiols, 50 µl of E. coli MG1655 or B. subtilis 168 were incubated as above for H₂O₂ production for 30–120 min (30 min was the optimum time for depletion of thiols, determined in preliminary experiments), and the total amount of reduced thiols was determined using fluorescent Measure-iT Thiol Assay Kit (InVitrogen/Molecular Probes) [35] (link) according to the manufacturer's instructions.