Ni Sepharose High Performance consists of highly cross-linked 6% agarose beads (34 µM) to which a chelating group has been immobilized and subsequently charged with Ni2+ ions. The chromatography medium provides very high binding capacity for histidine-tagged proteins and shows negligible leakage of Ni2+ ions.
Ni Sepharose High Performance is compatible with all commonly used aqueous buffers, reducing agents, and denaturants such as 6 M Gua-HCl and 8 M urea, as well as a range of other additives, and allows thorough procedures for cleaning the medium (Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products). It is stable over a broad pH range. This high chemical and physical stability and broad compatibility allows maintenance of biological activity and increases the yield of the puriﬁed product.
The good ﬂow properties and high resolution make Ni Sepharose High Performance the choice for high-performance puriﬁcations for the main characteristics of Ni Sepharose High Performance.
Ni Sepharose High Performance is supplied preswollen in 20% ethanol, in pack sizes of 25 and 100 mL, as well as in the convenient prepacked formats described later in this chapter.
Figure 1.Ni Sepharose High Performance precharged with Ni2+ for high-performance puriﬁcation of histidine-tagged proteins.
View Column packing and preparation for general guidelines for column packing.
This sample preparation procedure is applicable for all formats containing Ni Sepharose High Performance (Cell Lysis).
Adjust the sample to the composition and pH of the binding buffer by adding buffer, NaCl, imidazole, and additives from concentrated stock solutions; by diluting the sample with binding buffer; or by buffer exchange. To prevent the binding of host cell proteins with exposed histidines, it is essential to include imidazole at a low concentration in the sample and binding buffer (Optimizing purification of histidine-tagged proteins).
Pass the sample through a 0.22 µM or a 0.45 µM ﬁlter and/or centrifuge it immediately before sample application. Filtration is not necessary when using His MultiTrap HP. If the sample is too viscous, dilute it with binding buffer to prevent it from clogging; increase lysis treatment (sonication, homogenization); or add DNase/RNase to reduce the size of nucleic acid fragments.
Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 20 to 40 mM imidazole, pH 7.4. (The optimal imidazole concentration is protein dependent; 20 to 40 mM is suitable for many proteins.)
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4.
Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.45 µM ﬁlter before use. Use high-purity imidazole, as this will give a very low or no absorbance at 280 nm.
The optimal concentration of imidazole needed in the sample and buffer to obtain the best purity and yield differs from protein to protein. In the binding buffer, 20 to 40 mM imidazole is suitable for many proteins; 500 mM imidazole in the elution buffer ensures complete elution of the target protein.
As an alternative to elution with imidazole, lower the pH to approximately pH 4.5. (Metal ions will be stripped off the medium below pH 4.0.)
The column does not need to be stripped and recharged between each puriﬁcation if the same protein is going to be puriﬁed. Reuse of any puriﬁcation column depends on the nature of the sample and should only be performed with identical proteins to prevent cross-contamination. For more information on this topic and on cleaning and storage, refer to Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products.
Use the elution buffer as blank when measuring absorbance manually. If imidazole needs to be removed from the protein, use a desalting column (Desalting/buffer exchange and concentration). Low-quality imidazole will give a signiﬁcant background absorbance at 280 nm.
Ni Sepharose High Performance is compatible with reducing agents. However, we recommend removal of any weakly bound Ni2+ ions before applying buffer/sample that includes reducing agents. This can be accomplished by performing a blank run without reducing agents (see below). Do not leave or store Ni Sepharose High Performance with buffers that include reducing agents.
Leakage of Ni2+ from Ni Sepharose High Performance is low under all normal conditions. For very critical applications, leakage during puriﬁcation can be even further diminished by performing a blank run (as described below) before loading sample.