Can you specify what methods you have been trying?

Also, are you taking about the disulfide bond in protein?

Yun-Tzai Lee

I used several disulfide bond-reducing agents including 2-Mercaptoethanol.

No, there is a disulfide bond between two chemical compounds.

I want this bond to revert to its original state: two SH groups.

Dear Faezeh Ahrari

generally incubation at RT with strong reducing agents as DTT or TCEP (i do not like so much BME) at mM concentrations are able to broke disulfides, the problem is that if you are not working in anaerobic enviroment the SH can be reoxidezied and for S-S in short time once you remove the reducing agent.

Jusy a question: How do you monitor the disulfide integrity?

best

Manuele

Manuele Martinelli

Thanks for your answer

So, is it possible that in the presence of BME, this S_S bond is broken but re-oxidized and formed?

You can treat the small molecule with DTT or TCEP for couples of hours at room temperature or for 12-hrs at 4 degree C. I prefer TECP because it is a way stronger reducing agent than DTT, but you need to avoid using any phosphate buffer in the solvent.

Make sure you have sufficient amount of reducing agents (20~50-mM DTT or 0.5~1-mM TCEP) and buffer molecules to maintain solvent pH even if most reducing agents work with a wide pH range (for example, TCEP is useful at the range of pH 1.5-8.5). I will suggest to keep your buffer at lower pH, which can prevent disulfide bond formation after the reaction.

Lyophilize the sample solution right after the reaction is done. This step will remove the solvent which is a critical step to prevent from the re-oxidation.

Check the molecular weight using LC/GC mass spectrometry. If you don't want to heat up your molecule, use LC mass spectrometry.

Good luck!

Yun-Tzai Lee

Thank you for your answer