Two-Dimensional Gel Electrophoresis of all cellular proteins through an SDS gel can separate proteins having relatively large differences in molecular weight but cannot resolve proteins having similar molecular weights (e.g., a 30-kDa protein from a 31-kDa protein).

Dear Avinash,

Thank you for your reply. I always to 2-D SDS PAGE to confirm the purity of proteins along side with western blot. I'm actually concern about separation of proteins of similar molecular weights.

@Kaniz Fatema That's why I am saying , 2D gel electrophoresis is easy and best for identifying of two different molecular weight proteins but it can't separate two proteins having same molecular weight.

It will be quite challenging to separate these proteins based on their pIs, provided that these are experimental pI values and not theoretical ones. Check their hydrophobicity pattern, using one of the tools in Expasy. if they are different, you can use hydrophobic columns, such as Butyl-Sepharose column.

Good luck