If the non-binding and washing eluates have more protein than the actual high-imidazole eluate your problem is not elution, but binding. Access to the tag might be hindered by the structure of the protein (or it might be interacting with a charged patch on the surface of the protein, for instance), or the tag might have been cleaved off by a host protease.

Try Western blotting with an anti-His antibody to see if the tag is being cleaved off. If it isn't, then try doing the affinity purification in the presence of low concentrations of chaotropes (urea or GuHCl) to see if you can get that tag exposed.

Hope it helps!

You could try the batch purification process after extensive regeneration of the column with Nickel. It worked at my laboratory but using HIS-Select HF Nickel affinity Gel from Sigma.

What is the size of the column? Are the 16.9, 1.9, and 1 values a measurement of mass or concentration?

Thanks for your responses,

@Alejandro: We realized the western blot with an anti-His antibody , and we confirmed have found our protein at the size. So I think that the tag is exposed and not cleavec

@ Ahmed: Yes, I may be try

@ Omar: The column is 1 ml. 16.9 1.9 and 1 are the quantities measured in the total volume of sample passed, wash and eluate respectively

Emilie,

The positive Western blot only tells you that the tag has not been cleaved off; it says nothing about exposure/accessibility (SDS-PAGE and asorption onto a membrane will often change the conformation of the protein).

By the way, now I realize I misinterpreted completely your original post. You are saying that you loaded 19.9 ug but only 1.9+1 came out? Or are you saying that 19.9 ug passed through without binding?

Hi Alejandro,

Yes maybe my tag is not in a good conformation

But (and answer to your second question) : in quantity (µg)

in sample loaded: 210

in sample after passed trought the column: 16.9

in wash passed trought the column 1.9

in first elution fraction: 1

I hope I clarify