Determination of reduced cysteine residues

The number of reduced cysteines was determined using Ellman’s reagent (5,5’-dithiobis(2-nitrobenzoic acid)) (Ellman, 1959). Reactions were carried out under denaturing conditions in 8 M urea in the presence of 400 µM Ellman’s reagent and 5 µM ctPrp43 or ctPrp43-IDSB. The number of sulfhydryl groups was calculated by measuring the absorption at 412 nm using an Ultrospec 2100 pro UV/Vis spectrophotometer (GE Healthcare) and applying an extinction coefficient of 14290 M⁻¹ cm⁻¹.