English: Additional File 4 Ellman's Reagent to quantify Thiol reduction by NAC, DTT, and oxidation by Diamide. Figure Legend: DTT and NAC increase detection by Ellman's reagent. DTT (0.01, 0.05, 0.1, 0.5, 1.0, 5, 10 mM) and NAC (0.01, 0.05, 0.1, 0.5, 1.0, 5, 10 mM) were added to DTNB. The lowest concentration of NAC that was detectable to reduce the disulfide bond in DTNB was 2.0 × 10-9 M, well below what was used in our experiments with TGF-β. To these concentrations, increasing amounts of Diamide (0.1, 0.2, 1.0, 2.0, and 10 mM) were added and the ability to reoxidize DTNB was noted as absorbance at 410 nm. 10 mM diamide was not able to reoxidize DTNB at the highest concentrations of DTT, but was able to reoxidize 10 mM NAC treated DTNB. These data represent four redundant measurements from 3 independent studies. The line for 1.0 was left off the graph for simplicity of viewing. Method: Ellman's reagent (3,3'-dithio-bis(6-nitrobenzoic acid), DTNB) at 1 mmol in PBS with 10 mM EDTA was placed in a 96 well plate at 100 μl per well. Identical plates were made for measuring the reducing power of NAC and DTT under physiological conditions. Concentrations of 0, 0.01, 0.05, 0.1, 0.5, 1.0, 5, and 10 mM NAC and DTT were titrated against 0, 0.1, 0.2, 1.0, 2.0, and 10 mM diamide. The absorbance was checked on an ELx 808 at 410 nm using 570 nm as a correction for the absorbance of the plates. Results: NAC and DTT reduce disulfide bonds which is reversed by diamide. To confirm that the concentrations of NAC and DTT we used in these studies were sufficient to reduce disulfide bonds, we used Ellman's reagent to detect formation of free sulfhydryl residues. Treatment of Ellman's reagent with NAC or DTT induced a 0.98 ± 0.02 and 1.6 ± 0.1 molar ratio increase in free sulfhydryl groups, respectively. Diamide was able to re-oxidize the disulfide bond at equimolar concentrations of the reducing agents, to form a colorless compound. Complete oxidation was noted at 10-fold excess of diamide to NAC and all but the highest concentration of DTT.