I have purified a monomeric EGFP- His protein with a molecular weight of 30 kDa. But, instead of 30 kDa I am getting a thick band of 60 kDa in SDS-PAGE reducing gel. what is the possible reason for that?
Hi,
Did confirm your protein by western blot?.
If you could detect your protein either by His or GFP antibody then it will explain the protein you are getting is your target protein. If you have used BME in the loading Dye then the protein you are seeing mostly is not your target protein.
Kannan Balakrishnan , western confirmation is not required as the protein concentration is too high that the it appears green while eluted in the elution buffer and also after reading the literature I have got to know that it is very much possible to obtain a dimer in SDS-PAGE reducing gel if the interaction is strong. It is specially in the case if the proteins are membrane raft proteins.
- Badges
- Science method