Abstract
Thiol-based redox regulation is a posttranslational protein modification that plays a key role in many biological aspects. To understand its regulatory functions, we need a method to directly assess protein redox state in vivo. Here we present a simple procedure to determine protein redox state in a model plant Arabidopsis thaliana. Our method consists of three key steps: (i) redox fixation by rapidly freezing plant tissues in the liquid nitrogen, (ii) labeling of thiol groups with the maleimide reagent, and (iii) protein detection by Western blotting. The redox state of a specific or given protein can be discriminated by the mobility change on SDS-PAGE with high sensitivity. This method provides a novel strategy to dissect the working dynamics of the redox-regulatory system in plants.