Hi Hidayatullah,

It seems you're facing a challenging dimer that resists disruption. Since reducing agents like DTT or β-ME and heating haven't yielded results, consider exploring a few additional approaches.

• First, verify that you are using an optimal concentration of the reducing agent and allowing sufficient time for it to work before loading your sample onto the gel. You might also try increasing both the temperature and duration of heating the sample for 10-15 minutes while including the reducing agent, which could be beneficial.
• Additionally, adjusting the pH of your sample buffer may help, as some proteins exhibit greater stability at specific pH levels.
• You could also experiment with different detergents like SDS or chaotropic agents like urea or guanidine hydrochloride to ensure complete denaturation and disruption of intermolecular interactions.
• Also. You can run a control using a known monomeric form of your protein, which can provide insights into whether the dimer is really persisting under your current conditions or that band is something else?

Try these suggestions; I hope anyone of these will work for you. Good Luck.

Gaurav Chhetri Thanks for your suggestions