The Nickel-Nitrilotriacetic Acid (Ni-NTA) Kit provides the components necessary for 6 purifications of histidine (his) -tagged proteins expressed in
E.coli. The kit contains lysis, wash and elution buffers for purification under native conditions (i.e. without unfolding the protein). The buffers that have been optimized for native conditions are not sufficient for complete lysis of bacterial cells (usually
E.coli). Additional lysis steps like sonication can be employed to get complete lysis. For purifying proteins under denaturing conditions (i.e. unfolding the protein), a denaturing buffer is provided. Six columns prepacked with Ni-NTA resin are also provided. Since there is no way to close these columns, they are not reusable. The protocol book is quite comprehensive and allows a new user to understand the basics of protein expression and purification and thus, carry out the affinity chromatography with ease.
I have been using Ni-NTA chromatography for protein purification since 1999. I have purified a number of proteins using this technology. Though it’s good to get a hands-on-training with this kit, the final outcome in terms of purity and yield is not very encouraging. You would feel the need to standardize the protocols further with your own buffers. Too much loading of whole protein extract will result in leaching and the appearance of several other bands of proteins on a Coomassie-stained gel. Loading too little whole protein extract will result in non-specific binding and the appearance of non-specific bands. For large volumes, Ni-NTA bound to agarose beads (available in the form a slurry) can be obtained separately and packed in columns available from Qiagen. For smaller volumes, less slurry can be utilized. One should expect around 80% purity as determined by SDS-PAGE followed by Coomassie staining. Therefore, further purification steps are needed to increase the purity.
I would recommend this kit to beginners just for training purposes. It provides an affordable, easy-to-use, all-in-one kit for purifying his-tagged proteins which have been over-expressed in bacteria. Once the know how is developed, the researcher can move on to buying the individual components (available from Qiagen) and standardizing his/her own protocol based on the known properties of the protein to be purified.
For immunological purposes, it’s advised to remove the his-tag prior to using it for raising antibodies. Cloning of the desired gene can be done in vectors that allow the cleavage of his-tag after expression and purification.
This kit is worth its price and should be tried before moving onto actual purification experiments.