IMAC Sepharose® High Performance is an uncharged chromatography medium consisting of 34 µm beads of highly cross-linked 6% agarose to which a chelating group has been covalently coupled. This chelating group will be charged with suitable metal ions by the user, allowing the medium to selectively retain target proteins. The small bead size allows high chromatographic resolution with distinctly separated peaks containing concentrated material. The chromatography medium is highly compatible with a range of additives and is well suited to high-performance puriﬁcations that produce concentrated products in the eluate. Appendix 1 (Characteristics of Ni Sepharose®, Ni Sepharose® excel, TALON® Superflow, and uncharged IMAC Sepharose® products) shows the main characteristics of IMAC Sepharose® High Performance.
IMAC Sepharose® High Performance is supplied preswollen in 20% ethanol.
Fig 3.47. IMAC Sepharose® High Performance is supplied free of metal ions, enabling it to be used across a range of applications for purifying histidine-tagged as well as native proteins. It is available in 25 ml and 100 ml lab packs as well as prepacked HiTrap™ IMAC HP 1 ml and 5 ml columns.
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 IMAC Sepharose® High Performance.
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. 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.
Solutions of Zn2+ ions should have a pH of approximately 5.5 or lower to avoid solubility problems that arise at pH 6 or higher. Fe3+ ions should be immobilized at low pH (approximately pH 3.0) to avoid formation of insoluble ferric compounds.
Wash only with water, not buffer, prior to applying the metal ion solution, otherwise unwanted precipitation may occur.
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).
IMAC Sepharose® High Performance is compatible with reducing agents. However, we recommend removal of any weakly bound metal 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 IMAC Sepharose® High Performance with buffers that include reducing agents.
Leakage of metal ions is low under all normal conditions. For critical applications, leakage during puriﬁcation can be diminished by performing a blank run (as described below) before loading sample.
Use binding buffer and elution buffer without reducing agents.
For A280 measurements, use the elution buffers as blanks. If imidazole needs to be removed, use a desalting column (Chapter 11, Desalting/buffer exchange, and concentration). Low-quality imidazole will give a signiﬁcant background absorbance at 280 nm.
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