Puzzling ICP-ES Results, Metal Binding Protein?

Hi, there.

ICP-ES test was performed using my protein which was expressed and purified
from E.coli strain Origami(DE3), in order to find out whether it bond metal
or not. The results were puzzling.

Without His tag, the protein concentration was ~34 micromolar, while the Zn
was about ~17 micromolar, Fe was about ~8 micromolar, Cu was ~0.3
micromolar, Ni was ~0.4 micromolar. Other metals were negligible including
Co, Cr, Mg. Buffer were used as references.

In a separate test, the protein with His tag was ~47 micromolar, while Zn
was ~24 micromolar, Fe was ~9 micromolar, Cu was ~6 micromolar, Ni was ~4
micromolar. Other metals were negligible including Co, Cr, Mg. Buffer were
used as references.

Well, based on these results, I suspected my protein was a zinc binding
protein. However, I could not rule out other possibilities, especially Fe.

Have anyone performed ICP-ES before? Is it a reliable method?

Could a Zinc binding protein also bind Iron?

Are there any method available, other than in-cell NMR, to determine whether
my protein bind metal or not in vivo?

Thanks in advance!

Sincerely,

Pattis

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