Nitrilotriacetic acid (NTA), is a tetradentate chelating adsorbent developed by Hoffmann-La Roche that overcomes these problems. NTA (Fig. 3) occupies four of the six ligand binding sites in the coordination sphere of the nickel ion, leaving two sites free to interact with the 6xHis tag. NTA binds metal ions far more strongly than other available chelating resins and retains the ions under a wide variety of conditions, especially under stringent wash conditions. The unique, patented NTA matrices can therefore bind 6xHis-tagged proteins more tightly than IDA matrices, allowing the purification of proteins from less than 1% of the total protein preparation to more than 95% homogeneity in just one step.

Figure 4: Binding of His-tag with Ni-NTA groups.