Manual Purification of Histidine-Tagged Proteins using His GraviTrap™ TALON®

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

His GraviTrap™ TALON® columns are prepacked with 1 ml of TALON® Superflow medium. Each column provides simple manual purification of up to 15 mg of histidine-tagged proteins. The different purification steps are performed using gravity flow and require no further equipment (Fig 3.42). Large sample volumes can be applied in one go, and the histidine-tagged protein is effectively eluted in a small volume. Each package contains 10 prepacked columns, and each column is delivered in a package that converts into a column stand (Workmate). The plastic tray in the product package can be used to collect liquid waste. The columns are manufactured from biocompatible polypropylene, and special frits in each column protect the medium from running dry during purification. See Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose excel, TALON® Superflow, and uncharged IMAC Sepharose products) for the main characteristics of His GraviTrap™ TALON®.

 His GraviTrap TALON

Fig 3.41. His GraviTrap™ TALON® is a prepacked gravity-flow column for convenient manual purification of histidine-tagged proteins.

 

Schematic workflow for purifying histidine-tagged proteins on His GraviTrap TALON

Fig 3.42. Schematic workflow for purifying histidine-tagged proteins on His GraviTrap™ TALON.


Sample and buffer preparation

Refer to Purification using TALON® Superflow earlier in this chapter for a general procedure for sample and buffer preparation.

 

Purification

The protein binding capacity of the column is approximately 15 mg of histidine-tagged protein/ column (protein dependent). Connecting LabMate reservoir (see Ordering information) to the column increases convenience when handling volumes above 10 ml. This raises the loading capacity to approximately 35 ml in one run.

Prepare and equilibrate

  1. Cut off the bottom tip, remove the top cap, pour off excess liquid, and place the column in the Workmate column stand.
  2. If needed, mount LabMate on top of the column.
  3. Equilibrate the column with 10 ml of binding buffer. The frits protect the column from running dry during the run.

Load sample

  1. Add the sample. A volume of 0.5 to 35 ml is recommended.

After thorough cell disruption, it is possible to apply the unclarified sample directly to the column without experiencing clogging problems.

Wash

  1. Wash with 10 ml of wash buffer.

Elute

  1. Apply 3 ml of elution buffer, and collect the eluate.
  2. Under denaturing conditions, elute with 2 × 3 ml of elution buffer.

If using buffers that contain denaturing agents or viscous solutions, perform the purification at room temperature.

 

Application example

Simple and rapid protein purification using His GraviTrap™ TALON

His GraviTrap™ TALON® enables fast and easy manual purification of histidine-tagged proteins without the need of a purification system. Using a gravity-flow protocol, histidine-tagged green fluorescent protein (GFP-His) added to E. coli lysate was purified with His GraviTrap™ TALON® in less than 30 min. The recovery was calculated using absorbance measurements and was found to be 95%. Figure 3.43 shows the SDS-PAGE analysis of the first and second elution fractions. The results indicate a purity of ≥ 93% for the purified GFP-His protein.

SDS-PAGE analysis

Figure 3.43. SDS-PAGE analysis of elution fractions from a purification of GFP-His (Mr 28 000) added to E. coli lysate. The SDS-polyacrylamide gel (reducing conditions) was analyzed with ImageQuant TL software. The experiment was performed in triplicate. The first elution fraction contained 96% of the purified protein.

Materials

     
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