I am purififying one membrane transporter protein from SF9 cells. I can see glycosylation of my protein on SDS-PAGE. I tested Ni-agarose, Co-agarose, Zn-agarose and metal free agarose matixes. I see no binding with metal free agarose. Why?
If I understand the question correctly, you have a His Tag protein that binds Ni, Co, and Zn agarose but not agarose itself.
If this is correct, then the His Tag is binding metals as it is designed to do.
if you do not know what is the purpose of using metal free agarose, maybe you should ask to the person that design the experiment. I can only guess that it is a control experiment to verify non-specif binding.
histidine residues form coord. complexes with transitional metals. Why do you used metal containing gel?
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