Protein Redox Modification as a Cellular Defense Mechanism against Tissue Ischemic Injury

Figure 3

Analysis of reversible cysteine redox modifications. What is shown is a one of the popular methods generally called a “biotin switch” assay, which involves alkylating the free thiol groups (step 1), reducing the modified cysteine residues using specific reductant for each oxidation product (step 2), and relabeling of the newly generated free thiol groups using biotin conjugated probes (step 3). Following biotinylation, the samples can be further analyzed by either western blot (step 5) or affinity purification (step 4). Note that protein sulfenic acids can be directly labeled by dimedone conjugated biotin probes as described in the text.