I purified my enzyme with 250 mM imidazole by ni-agarose column, my enzyme has 2 metal in structure ( Ca and Zn). Is it necessary to add this metal into buffer dialysate?
It is hard to tell whether your metal ions are stripped by the Ni affinity column. It is always possible, especially for Zn which can bind to the Ni sites on the column. In general Ca binds rather weakly to most its sites in where it is a catalyst (except things like calmodulin and other specific Ca binding proteins where it is really structural) but if it bind tightly the good news is that its less likely to be stripped by the Ni column. Imidizole is a good Zn chelator so it too can cause problems with stripping your Zn, depending upon how it binds to your protein. Zn sulphur bonds, in proteins like Zn fingers tend to be kinetically stable (unless you unfold the protein), while in enzymes where its coordination shell is not fully filled by protein ligands it can be less stable (carbonic anhydrase). It is always possible to add the metals to the dialysis buffer. The key question is what are your buffer components. If you need reducing agents then with Zn you probably want to use beta mercaptoethanol or TCEP since DTT and Zn form a complex that will tend to precipitate. It should be noted that EDTA will bind both Ca and Zn so if you have it in your buffer and the protein-metal complex is weak you will strip your metals. Basically, the two key questions are whether the buffer components have a higher affinity for the protein or whatever is in the buffer, and whether the kinetic of release from the protein are such that the metals can be efficiently trapped by the Ni column or buffer components. Finally, you should remember that for most Ni affinity resins, particularly as they age with use, Nickle is released and your samples will have varying degrees of Ni contamination, which itself could affect the Zn content of your protein.
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