The redox state of cysteine thiols is critical for protein function. Whereas 
cysteines play an important role in the maintenance of protein structure through 
the formation of internal disulfides, their nucleophilic thiol groups can become 
oxidatively modified in response to diverse redox challenges and thereby 
function in signalling and antioxidant defences. These oxidative modifications 
occur in response to a range of agents and stimuli, and can lead to the 
existence of multiple redox states for a given protein. To assess the role(s) of 
a protein in redox signalling and antioxidant defence, it is thus vital to be 
able to assess which of the multiple thiol redox states are present and to 
investigate how these alter under different conditions. While this can be done 
by a range of mass spectrometric-based methods, these are time-consuming, 
costly, and best suited to study abundant proteins or to perform an unbiased 
proteomic screen. One approach that can facilitate a targeted assessment of 
candidate proteins, as well as proteins that are low in abundance or 
proteomically challenging, is by electrophoretic mobility shift assays. 
Redox-modified cysteine residues are selectively tagged with a large group, such 
as a polyethylene glycol (PEG) polymer, and then the proteins are separated by 
electrophoresis followed by immunoblotting, which allows the inference of redox 
changes based on band shifts. However, the applicability of this method has been 
impaired by the difficulty of cleanly modifying protein thiols by large PEG 
reagents. To establish a more robust method for redox-selective PEGylation, we 
have utilised a Click chemistry approach, where free thiol groups are first 
labelled with a reagent modified to contain an alkyne moiety, which is 
subsequently Click-reacted with a PEG molecule containing a complementary azide 
function. This strategy can be adapted to study reversibly reduced or oxidised 
cysteines. Separation of the thiol labelling step from the PEG conjugation 
greatly facilitates the fidelity and flexibility of this approach. Here we show 
how the Click-PEGylation technique can be used to interrogate the redox state of 
proteins.