Ni-NTA Agarose From Qiagen

The Nickel Nitrilo-triacetic Acid (NTA) is a Qiagen-patented resin which offers affinity purification of 6Xhis-tagged proteins expressed as recombinant proteins from sources like E. coli, insect (baculovirus), yeast or mammalian expression systems. This technique makes use of the affinity of multiple histidine residues for nickel (Ni).

To prepare your recombinantly expressed proteins, lyse the cells to release or solublize the expressed protein and centrifuged to yield a clear lysate. The purify your recombinant protein, mix the lysate with the resin by either stirring/shaking the lysate with the Ni-NTA resin for about an hour or by passing the lysate 2-3 times over a column packed with the Ni-NTA resin (i.e. either batch or column method, respectively). A 3 ml volume of the Ni-NTA resin is usually sufficient to purify protein from 1 L of E. coli culture. About 100 ul of resin is sufficient to purify and visualize recombinant protein by SDS-PAGE from 3 ml culture of E. coli. For elution of the affinity bound His-tagged protein, a high concentration of imidazole, an histidine analog, is used.

The Ni-NTA resin can be used to purify all His-tagged proteins irrespective of whether the tag is attached to N-terminal or C-terminal. Of course, if the tag is not exposed or buried in the native folded structure of the proteins, it will be difficult to purify the native protein. In this case, the protein can be purified by denaturing the protein with 6M guanidine hydochloride or 8M urea to expose the tag; this method is also used when expressed proteins form inclusion bodies. The presence of high concentrations of guanidine or urea does not affect the binding of the protein to the resin. Rather, it decreases the chances of protease digestion and breakdown of the recombinant proteins. Denatured protein can be refolded on the column by gradual removal of the denaturant followed by washes to remove any traces of denaturants. Finally, protein is eluted with imidazole either by gradient of 0-250 mM concentration or by empirically determined concentrations. At this stage, the protein is generally sufficiently pure to carry out studies or assays.

The Ni-NTA agarose resin is supplied as 50% slurry with ethanol; it is washed with de-ionized water to remove the ethanol and equilibrated with buffer prior to use. It can also be used for protein mini-preps as well as pull-down assays using microfuge tubes or mini spin columns. In my experience, the resin can withstand high centrifugal force without damage can be reused number of times. The resin can be recharged after each purification following the recharging protocol in the user manual. Although the protocol may look tedious, it is easy and quick.

Ni-NTA has really made the tedious process of protein purification child’s play. It excludes the preparative techniques like gel permeation or ammonium sulfate precipitation. Packing a Ni-NTA column is much easier and does not require any precautions as in case of other chromatographic columns. Special care must be taken with columns like gel filtration, ion-exchange, etc. in order to avoid the formation of even the tiniest of bubbles which might interfere with protein resolution and the purification profile; this makes these techniques extremely cumbersome in comparison to Ni-NTA chromatography.

The purity of proteins may vary from 70% to 90% or even more. Variations depend upon the batch of resin used and upon factors like the amount of resin used, the height to width ratio of the column, the amount of expressed protein present and the volume of the cell lysate. Use of a protease inhibitor cocktail may immensely affect the protein yield and purity by reducing protein degradation.

The Qia-expressionist handbook provided with the resin is a good reference for optimizing purification protocols and troubleshooting.