I am using Swanella bacteria (gram negative) to purify cytochrome (a periplasmic protein, C type heme, homodimer). I put 10 Histag at the C terminal region, because N terminal region have signaling sequence for periplasm. I tried to load cell lysate to the Ni coulmn applying different concentration of Imidazole (5mM-70mM Imidazole, 20mM Tris Base, 05M NaCl, pH=8.1), most of the protein don't bind to the column which I confirmed by checking flow through. Would you please suggest me whats happening here ?
Is it carboxypeptidase enzyme which cleaving Histag from C terminal, that's why protein is not binding to the column? or any other reason.
I appreciate your valuable suggestions. Thanks