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Extended reaction time Reagent Preparation Prepare 4 N NaOH Add 15 mL water to flask Add 5.2 mL 50% NaOH (19.4 N) to 25 ml volumetric flask Mix Cool to room temperature Bring volume to 25 mL with water Store in a plastic bottle at room temperature Add 4 g NaOH pellets to 25 ml volumetric flask Add 20 mL water volumetric flask Mix until dissolved Cool to room temperature Bring volume to 25 mL with water Store in a plastic bottle at room temperature Prepare calibrators in a 2 to 1 MPA:Assay Buffer Solution 0 uM GSSG - MPA:Assay Buffer 5 uM GSSG - 25 uL calibrator concentrate in 975 uL MPA:Assay Buffer Solution 10 uM GSSG - 50 uL calibrator concentrate in 950 uL MPA:Assay Buffer Solution Note: Calibrators will be treated and assayed the same as the samples except they don't need to be frozen