In my experience, it will be difficult to identify the labelling by different running behaviour in SDS-PAGE analyses, at least in a direct manner. What you can do is to label the free thiols (i.e. those that have not been labeled with IAA) with methoxypolyethylene glycol maleimid-5000 (MalPEG). You should then see a shift of about 10 kDa for each free thiol derivatized with MalPEG compared to your non-derivatized protein in tris-tricine gels. If you now derivatize a batch with MalPEG but no IAA labelling and one with IAA labelling, you should be able to determine exactly how many of your cysteines are derivatized with MalPEG by the shift of 10 kDa. Here you can find the procedure of the MalPEG Gel-Shift Assay described in detail:

https://www.sciencedirect.com/science/article/pii/S0006291X1301807X?via%3Dihub

https://www.frontiersin.org/articles/10.3389/fmicb.2018.01610/full

Another possibility would be to detect free thiols using DTNB, as you have already mentioned before, by creating a calibration line with free thiols and incubating your protein with DTNB as well. However, you will probably need to precipitate your protein to remove excess DTNB. A similar approach was done by Niu et al 2018, maybe this will help you (https://www.jbc.org/content/293/7/2523).

Free radical spin probes are used for electron spin (aka paramagnetic) resonance (ESR/EPR). So why not put your sample into the machine and check for the signal? It should be sensitive to radical scavengers like ascorbic acid or chromium oxalate (CROX).

If you want to detect spin labels after electrophoresis, you'd have to be careful that the label is not destroyed. After all, polymerisation of polyacrylamide is by radicalic reactions. At the very least, before use keep the gel o/n in the fridge in a humid chamber so that most of those chemicals have a chance to decay. You also need to think about the sample buffer, thiols probably should be kept out.

Dear HIroyasu

If you protein is not very big and number of Cysteines limited you can bind it with AMS (thermo cod A-485) and you will observe the shift of about 850Da for each cisteine in SDS-page.

Of course the analysis is simple if you have just 2 cysteines involved in 1S-S bond while it become more complex if you have many cysteines of the MW of your protein is very high and the 1,7Kda shift is not observable.

This is useful if you would like to follow the oxidation state of the cysteines.

AMS is fluorescent, therefore you can use also fluorescence as detection tool but in this case you need to perform dialysis before read the fluorescence to remove the unbounded AMS.

Ciao

Manuele