Ni Sepharose excel consists of 90 µm highly cross-linked agarose beads, to which a chelating ligand has been coupled. The ligand is precharged with nickel ions that are exceptionally strongly bound, thus enabling direct loading of samples that usually cause stripping of nickel from conventional IMAC media. For example, the nickel ions remain bound even after 24 h of incubation in 10 mM EDTA.
Extensive and time-consuming sample pretreatment procedures, for example, buffer exchange by dialysis in combination with concentration procedures, can be avoided. Examples of samples that often cause stripping problems are cell culture supernatants containing secreted histidine-tagged proteins from eukaryotic cells, such as insect cells or CHO cells.
The ﬂow properties of Ni Sepharose excel make it excellent for puriﬁcations in all scales and allow loading of large sample volumes. Due to low target protein concentrations, several liters of sample often need to be processed when purifying secreted proteins.
See Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products) for the main characteristics of Ni Sepharose excel.
Figure 1.Ni Sepharose excel for puriﬁcation of histidine-tagged proteins secreted into eukaryotic cell culture supernatants; it is available in 25 mL, 100 mL, and 500 mL volumes.
Ni Sepharose excel is supplied preswollen in 20% ethanol. Suitable columns for packing are Tricorn™, XK, and HiScale™ columns. The column packing procedure differs from the general procedure for other chromatography media as described in Appendix 6 (Column packing and preparation). Follow this recommended packing procedure below for Ni Sepharose excel.
If the puriﬁcation is to be performed at a high ﬂow rate, HiScale columns are recommended.
Make sure no air has been trapped under the column bed support.
Avoid trapping air bubbles under the adapter or in the inlet tubing.
1 Should be at least 133% of the ﬂow rate to be used during subsequent chromatographic procedures. If this cannot be obtained, use the maximum ﬂow rate your pump can deliver. This should also give a well-packed bed.
Before sample loading, whole cells must be removed by, for example, centrifugation, otherwise clogging of the column may occur. When using HisTrap excel columns, no further clariﬁcation is needed. When using Ni Sepharose excel packed in other columns, it is recommended to also ﬁlter the sample through a 0.45 µm ﬁlter to remove cell debris and/or other particulate material.
Adjust the sample to the composition and pH of the binding buffer by adding buffer and NaCl from concentrated stock solutions or by diluting with binding buffer. For optimal binding, it is not recommended to include imidazole in sample and binding buffer. It is recommended to perform binding at neutral pH. However, successful puriﬁcation has routinely been observed with binding performed at a pH as low as 6.0. Due to the precipitation risk, avoid using strong bases or acids for pH adjustments.
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, which gives essentially no absorbance at 280 nm.
It is not recommended to include imidazole in sample and binding buffers.
Optimal imidazole concentration during wash is sample dependent. See Chapter 4 (Optimizing puriﬁcation of histidine-tagged proteins) for further information.
Puriﬁcation at low temperatures increases the sample and buffer viscosity, leading to increased back pressure. If necessary, decrease ﬂow rate.
For A280 measurements, use the elution buffers as blanks. If imidazole needs to be removed, use a desalting column (see Chapter 11, Desalting/buffer exchange and concentration). Low-quality imidazole will give a signiﬁcant background absorbance at 280 nm.
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