Thioredoxin redox western analysis.

Increasing evidence suggests that compartment-specific changes are important in redox signaling and control. Examination of thiol/disulfide redox changes in thioredoxin (Trx) family members including Trx1 in cytoplasm and nucleus and Trx2 in mitochondria should aid in the understanding of compartmentalized redox signaling mechanisms. Methods to quantify redox states of both Trx1 and Trx2 by redox western analysis and to further calculate redox potential using the Nernst equation are described in this unit. The procedures to measure redox states of Trx1 and Trx2 consist of three parts, derivatization, redox western blotting, and calculation of redox potentials. Derivatization of proteins with thiol-reactive reagents prior to redox western blotting prevents artifactual oxidation of protein thiols and allows separation of the reduced forms from oxidized forms of the protein. Consequently, the redox western analysis of Trx provides a convenient tool to estimate the redox state of different compartments and improve the understanding of redox signaling.