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.
The protein binding capacity of the column is high (approx. 40 mg histidine-tagged protein/column); however, the value is protein dependent.
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.
Use binding buffer and elution buffer without reducing agents.
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 (Mr ~ 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.