The Ni-NTA (nickel-nitrilotriacetic acid) protein purification system is a wonderful technique for affinity purification of recombinant proteins expressed with a (His)n tag at either the (N- or C-) terminus of recombinant proteins. Ni-NTA superflow columns come in packs of 12 or 144. Each polypropylene column contains 1.5 ml of Ni-NTA Superflow resin which is capable of binding up to 30 mg of protein. One of the benefits to these prepacked columns is that they allow you to move ahead with protein purification without having to take the time to pack and maintain the columns. However, the columns cannot be reused as there is no screw cap to close the columns after use.
Before the first use, the columns should be washed with 4-5 column volumes (6-7 ml) of the buffer in which the protein is kept. The tip of the column should be cut first and then the airtight cap should be removed to allow the gravity flow. Although there is no difference in the flow rate if the steps are reversed. Care should be taken not to allow any air bubbles to become trapped inside the slurry as it makes the flow rate extremely slow. The column can hold up to 10 ml of the buffer/protein. The blue color of the resin is due to the charged nickel ions; if the color has changed due to excessive loading, either the column should be recharged or a larger amount of slurry should be used.
I have used these columns for the past year, but have been using Ni-NTA affinity purification for the past 7 years. I used these columns to purify proteins of different molecular weights (17 kDa to 83 kDa). I have successfully purified a protein (80% as determined after Coomassie staining) with 8 histidine residues at the C-terminus. Usually a hexa-histidine tag is present in proteins purified with Ni-NTA affinity chromatography. I have found that reloading of the first flow through collected after loading whole cell extracts increases the overall yield of the purified protein. Caution should be exercised in choosing the protease inhibitors used during protein extraction. EDTA-containing protease inhibitors would chelate the Ni ions and prevent the tagged protein from binding. I used EDTA-free protease inhibitor cocktail tablets from Roche with success.
The only factor of concern for me was the extremely slow flow rate under gravity. For faster flow rates, these columns can be used with vacuum manifolds and other automated systems available from Qiagen (BioRobot Protein LS System or BioRobot 3000 workstation), but I have not used these systems myself. Automated procedures would allow purification of 24 proteins simultaneously. Due to the sturdy composition of the slurry (6% highly crosslinked agarose), higher flow rates can be achieved.
Overall, these prepacked columns are a preferred choice for affinity purification of proteins with significant yield and purity.