Purification of Histidine-Tagged Proteins using TALON^® Superflow

TALON^® Superflow consists of highly cross-linked agarose beads with an immobilized chelating group precharged with Co²⁺ ions.

The medium binds polyhistidine-tagged proteins with high selectivity and exhibits a reduced affinity for host proteins, giving lower background compared with other IMAC media. The medium is compatible with many aqueous buffers, denaturants, and a range of other additives (Appendix 1, Characteristics of Ni Sepharose, Ni Sepharose excel, TALON^® Superflow, and uncharged IMAC Sepharose products). Avoid using DTT (dithiothreitol), DTE (dithioerythritol), and TCEP (TRIS (2-carboxyethyl) phosphine) with TALON^® Superflow products. Protein binding capacity will decrease rapidly if used. The medium is suitable for batch purification, and can also be used for packing into liquid chromatography columns such as Tricorn or XK columns. The medium usually works well with protocols designed for Ni²⁺-based IMAC columns.

Sample preparation

This sample preparation procedure is applicable for all formats containing TALON^® Superflow. 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.

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 HiTrap TALON^® crude and His GraviTrap. If the sample is too viscous, dilute it with binding buffer; increase lysis treatment (sonication, homogenization); or add DNase/RNase to reduce the size of nucleic acid fragments.

Buffer preparation

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.

Nonspecific binding of proteins due to electrostatic interactions can be decreased by increasing the NaCl concentration up to 500 mM.

The imidazole concentration required for wash and elution is protein dependent. Chapter 4 (Optimizing purification of histidine-tagged proteins) for further information.

Column packing

TALON^® Superflow is supplied preswollen in 20% ethanol. Prepare a slurry by decanting the 20% ethanol solution and replacing it with distilled water in a ratio of 75% settled medium to 25% distilled water.

1. Assemble the column (and packing reservoir if necessary).
2. Remove air from the end-piece and adapter by flushing with water. Make sure no air has been trapped under the column bed support. Close the column outlet leaving the bed support covered with water.
3. 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.
4. If using a packing reservoir, immediately fill the remainder of the column and reservoir with water. Mount the adapter or lid of the packing reservoir and connect the column to a pump. Avoid trapping air bubbles under the adapter or in the inlet tubing.
5. Open the bottom outlet of the column and set the pump to run at the desired flow rate. This should be at least 133% of the flow rate to be used during subsequent chromatographic procedures. However, the maximum flow rate is typically employed during packing.

For subsequent chromatography procedures, do not exceed 75% of the packing flow rate.

10.  Maintain the packing the flow rate for at least 3 bed volumes after a constant bed height is reached. Mark the bed height on the column.
11.  Stop the pump and close the column outlet.
12. If using a packing reservoir, disconnect the reservoir and fit the adapter to the column.
13. With the adapter inlet disconnected, push the adapter down into the column until it reaches the mark. Allow the packing solution to flush the adapter inlet. Lock the adapter in position.
14.  Connect the column to a pump or a chromatography system and start equilibration. Re-adjust the adapter if necessary.

For subsequent chromatography procedures, do not exceed 75% of the packing flow rate.

Purification
Purification procedure for a packed column

1. If the column contains 20% ethanol, wash it with 5 bed volumes of distilled water. Use a flow velocity of 50 cm/h to 100 cm/h. Appendix 8 for information on converting flow velocity (cm/h) to volumetric flow rate (mL/min).
2. Equilibrate the column with 5 to 10 bed volumes of binding buffer. Recommended flow velocity: 150 cm/h.

In some cases, a blank run is recommended before final equilibration/ sample application. See below for blank run.

3. Apply the pretreated, filtered, or centrifuged sample.
4. Wash with wash buffer until the absorbance reaches the baseline.
5. Elute with elution buffer using a step or linear gradient. For step elution, 8 bed volumes of elution buffer are usually sufficient. A shallow gradient, for example a linear gradient over 20 bed volumes, may separate proteins with similar binding strengths.

Blank run:
Leakage of Co²⁺ from TALON^® Superflow is low under all normal conditions. For very critical applications, leakage during purification can be even further reduced by performing a blank run before loading sample.

1. Wash the column with 5 column volumes of distilled water.
2. Wash with 5 column volumes of elution buffer.
3. Equilibrate with 10 column volumes of binding buffer.