Your protein is either misfolded/aggregated or the his tag is chewed off/never translated. You could try detergents etc. To improve the state of the protein but I would also doubl check sequence for early termination (assuming cterm) or protelysis. Artem On Jan 15, 2012 12:23 PM, "Theresa H. Hsu" <[email protected]> wrote:
> Hi all > > I have a His-tagged soluble protein (8 His residues added to 90 kDa > protein) that do not bind to IMAC column based on flowthrough showing up > with Western blott. Do you have suggestions to improve the binding? > > Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH > to 8.0. > > Thank you. > > Theresa >
