Procedure of diagonal electrophoresis I am learning protein sequencing and it seems that diagonal electrophoresis is a common method in the identification of disulphide bonds (S-S) exist between polypeptides in a protein. **Questions:** 1. After horizontal electrophoresis, we need to cleave the S-S bond using performic acid. According to Wiki, > Its strong oxidizing properties are used for cleaving disulfide bonds in protein mapping. However, from what I have learnt in SDS-PAGE, 2-mercaptoethanol OR dithiothreitol are commonly used for cleaving S-S bonds, may I ask why do we need to use formic acid specifically here? 2. the first electrophoresis is done horizontally and the second is done vertically (bottom to top). May I ask whether we can do it vertically then horizontally instead? It seems to me that it should be OK since there doesn't exist any disruption to the proteins but I would like to confirm. Thank you.

You probably don’t _need_ to oxidize the disulfide bonds rather than reduce them. Performic acid has some potential advantages:

* The oxidation products can’t spontaneously reform a disulfide bond whereas the reduction products from a thiolate reductant can.
* Oxidation by performic acid causes a significant mass change for the cysteine residues which can be useful for downstream mass spec. This article states that complete oxidation of the cystine to cysteic acid occurs (-SO3-), which is a molecular mass change of 48 u. With BME or DTT reduction, you are left with a thiol or thiolate.
* These oxidation products are also negatively charged which can increase mobility in PAGE and possibly aid separation.