I have expressed a plant protein in E.Coli. This recombinant protein is His-tagged. Its size is 27KDa. Now when I purify my protein and run it in SDS-PAGE I have two bands very close to each other at about 27 KDa. I cut both of them out and sent them for peptide mass fingerprinting. Both of them turned out to be my protein. I have attached a photo of my purified protein in SDS-PAGE. Lane 5, 6, 7 are my purified protein elutions at 100-250mM imidazole. The lower band is lighter than the upper band.
I was wondering why this size difference? It cannot be a dimer as I heat my samples in SDS-loading buffer with DTT. Any suggestions on how is it possible to get the same protein in two close but different sizes, will be highly appreciated. I tried looking this kind of problem in papers but couldn't come across one.
Also I ran a native gel and I got one band at about 73 KDa and another about 65 KDa. They had quite a distance than what they appear in SDS-PAGE.
Edited by Bioscientist, 23 May 2013 - 07:46 PM.