Gravity-flow Purification using His GraviTrap™ and His GraviTrap™ Kit

His GraviTrap™ columns are designed for fast and simple purification of histidine-tagged proteins using gravity flow. Both clarified and unclarified sample can be applied to the column. The column is prepacked with Ni Sepharose^® 6 Fast Flow. Special column frits protect the chromatography medium from running dry during purification. A typical purification run on His GraviTrap™ is performed in approximately 20 min (depending on sample volume and viscosity of the solutions).

His GraviTrap™ columns are delivered in a package that can be converted into a column stand to simplify purification. LabMate PD-10 Buffer Reservoir can be connected to the columns for convenient handling of sample volumes above 10 mL. For optimal performance, use His GraviTrap™ with buffers prepared from His Buffer Kit.

Figure 3.21. His GraviTrap™ connected to LabMate PD-10 Buffer Reservoir for convenient equilibration, sample application, and wash.

The benefits of His GraviTrap™ and His Buffer Kit are combined in His GraviTrap™ Kit, which contains two packs of His GraviTrap™ and one pack of His Buffer Kit. His GraviTrap™ Kit contains columns and buffers for 20 purifications. Figure 3.22 for a summary of the purification procedure using His GraviTrap.

Figure 3.22. Purifying histidine-tagged proteins with His GraviTrap™ is a simple and quick procedure.

Sample and buffer preparation

Refer to Purification using Ni Sepharose^® 6 Fast Flow earlier in this chapter for a general procedure for sample and buffer preparation.

For direct loading of an unclarified sample, it is critical to obtain good cell lysis in order to avoid problems with back pressure. Apply the unclarified lysate on the column directly after preparation.

If the sonicated or homogenized unclarified cell lysate is frozen before use, precipitation and aggregation may increase. New sonication of the lysate can then prevent clogging the column.

Purification

1. Cut off the bottom tip, remove the top cap, pour off excess liquid, and place the column in the Workmate column stand. If needed, mount LabMate (funnel) on top of the column.
2. Equilibrate the column with 10 mL of binding buffer. The frits protect the column from running dry during the run.
3. Add 0.5 to 35 mL of the prepared sample.

The protein binding capacity of the column is high (approx. 40 mg histidine-tagged protein/column); however, the value is protein dependent.

4. Wash with 10 mL of binding buffer.
5. Apply 3 mL of elution buffer and collect the eluate. Under denaturing conditions, elute twice with 3 mL of elution buffer.

If you use buffers containing denaturing agents or viscous solutions, perform the purification at room temperature.

Ni Sepharose^® 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 His GraviTrap™ columns with buffers that include reducing agents.

Leakage of Ni²⁺ from Ni Sepharose^® 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.

Blank run:

Use binding buffer and elution buffer without reducing agents.

1. Wash the column with 5 column volumes of distilled water (to remove the 20% ethanol).
2. Wash with 5 column volumes of elution buffer.
3. Equilibrate with 10 column volumes of binding buffer.

Application example

Rapid purification of a high-molecular-weight histidine-tagged protein using His GraviTrap

His GraviTrap, prepacked with Ni Sepharose^® 6 Fast Flow, allows quick and simple purification of histidine-tagged proteins without the need for a pump or purification system. A single column allows purification of approximately 40 mg of protein in as little as 20 to 25 minutes. Large volumes of clarified or unclarified samples can easily be applied, and the purified protein can be eluted in a small volume, resulting in a highly concentrated target protein.

In this example, 20 mL of a clarified E. coli JM109 lysate containing (His)₁₀-TRX-P450 (M_r ~ 130 000) was purified in just 25 minutes and analyzed by SDS-PAGE and Western blot (Figure 3.23A and B). SDS-PAGE analysis shows three major protein bands in the eluted fractions. Western blot analysis and N-terminal sequencing (data not shown) confirm that each of the three bands in the eluates contains a histidine tag. The low-molecular-weight bands are truncated forms of the histidine-tagged target protein.

Figure 3.23. (A) SDS-PAGE and (B) Western blot of (His)10-TRX-P450 purified using His GraviTrap™ column.