Ni Sepharose 6 Fast Flow consists of 90 µM beads of highly cross-linked agarose, to which a chelating ligand has been immobilized and subsequently charged with Ni2+ ions. The ligand density of Ni Sepharose 6 Fast Flow ensures high binding capacity, and the chromatography medium shows negligible leakage of Ni2+ ions. The high ﬂow rate property of the Sepharose 6 Fast Flow matrix makes it well-suited for scaling-up but also for gravity-ﬂow purposes. In addition, the medium is compatible with a wide range of additives commonly used in the puriﬁcation of histidine-tagged proteins. Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products) for the main characteristics of Ni Sepharose 6 Fast Flow.
Figure 3.5.Ni Sepharose 6 Fast Flow is designed for scaling up puriﬁcation of histidine-tagged proteins.
Ni Sepharose 6 Fast Flow is supplied preswollen in 20% ethanol, in pack sizes of 5, 25, 100, and 500 mL1, as well as in convenient prepacked formats as described later in this chapter.
1 Larger quantities are available. Contact your local representative for more information.
See instructions supplied with the product, or refer to Appendix 6 (Column packing and preparation) for general guidelines for column packing.
This sample preparation procedure is applicable for all formats containing Ni Sepharose 6 Fast Flow. See Cell lysis earlier in this chapter for a general description.
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 histidine, it is essential to include imidazole at a low concentration in the sample and binding buffer (Chapter 4, 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 HisTrap FF crude, His GraviTrap, or His MultiTrap FF. 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.
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 tagged proteins to prevent cross-contamination. For more information on this topic and on cleaning and storage, refer to Appendix 1 (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 (Chapter 11, Desalting/buffer exchange and concentration). Low-quality imidazole will give a signiﬁcant background absorbance at 280 nm.
Ni Sepharose 6 Fast Flow 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 store Ni Sepharose 6 Fast Flow with buffers that include reducing agents.
Leakage of Ni2+ from Ni Sepharose 6 Fast Flow 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.
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