Purification of Histidine-Tagged Proteins Secreted into Eukaryotic Cell Culture Supernatants Using Ni Sepharose® Excel

Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

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.

Ni Sepharose excel

Fig 3.31. 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.

Column packing

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.

  1. Allow the medium slurry to sediment for 3 h.
  2. Add enough 20% ethanol to achieve a 1:1 ratio of settled medium and overlayed 20% ethanol.
  3. Assemble the column (and packing reservoir if necessary).

If the purification is to be performed at a high flow rate, HiScale columns are recommended.

  1. Remove air from the end-piece and adapter by flushing with 20% ethanol.

Make sure no air has been trapped under the column bed support.

  1. Close the column outlet leaving the bed support covered with 20% ethanol.
  2. Resuspend the medium and pour the slurry into the column in a single continuous motion. Pouring the slurry down a glass rod held against the column wall will minimize the introduction of air bubbles.
  3. Allow the medium slurry to sediment for at least 3 h.
  4. If using a packing reservoir, disconnect the reservoir and fill the remainder of the column with 20% ethanol.
  5. Mount the adapter and connect the column to a pump.

Avoid trapping air bubbles under the adapter or in the inlet tubing.

  1. Immediately open the bottom outlet of the column and set the pump to run at the desired flow rate1.
  2. Maintain packing flow rate for at least 3 column volumes after a constant bed height is reached.
  3. Stop the pump and close the column outlet.
  4. With the adapter inlet disconnected, quickly push the adapter down into the column until it reaches the bed surface and then a further 3 to 4 mm into the medium bed. Lock the adapter at this level.
  5. Connect the column to a pump or a chromatography system and start equilibration. Readjust the adapter if necessary.

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.

Sample preparation

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.

Buffer preparation

Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4
Wash buffer: 20 mM sodium phosphate, 0.5 M NaCl, 0 to 30 mM imidazole, pH 7.4
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, 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.


  1. If the column contains 20% ethanol, wash it with 5 column volumes of distilled water. Use a flow velocity of 50 to 100 cm/h. Refer to Appendix 8 (Converting from flow velocity to volumetric flow rates) for flow rate calculations.
  2. Equilibrate the column with at least 5 column volumes of binding buffer at a flow velocity of 150 to 600 cm/h.
  3. Load the sample at a flow velocity of 150 to 600 cm/h.
  4. Wash with 20 column volumes of wash buffer at a flow velocity of 150 cm/h.
  5. Elute with elution buffer using a one-step procedure. Five column volumes of elution buffer is usually sufficient. Alternatively, a linear elution gradient (10 to 20 column volumes) may give higher purity, at the expense of lower target protein concentration in eluted fractions. Use a flow velocity of 150 cm/h.

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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