Rapid Screening of Histidine-Tagged Proteins using His SpinTrap™ TALON®

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

His SpinTrap™ TALON® columns are excellent for screening of expression levels and purification conditions prior to scale up. The columns are designed for use in a microcentrifuge. Figure 3.40 shows the rapid purification procedure using His SpinTrap™ TALON®, taking approximately 10 min. The prepacked columns enable highly reproducible column-to-column results in yield and purity.

Each column contains 100 µl of TALON® Superflow, enough for purifying up to 1 mg of histidine-tagged protein. See Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose® excel, TALON® Superflow, and uncharged IMAC Sepharose® products) for the main characteristics of His SpinTrap™ TALON.

His SpinTrap TALON spin column

Fig 3.39 His SpinTrap™ TALON® is a single-use spin column for high-purity purification of histidine-tagged proteins.

 

Purifying histidine-tagged proteins with His SpinTrap™ TALON<sup>®</sup> simple four-stage procedure

Fig 3.40. Purifying histidine-tagged proteins with His SpinTrap™ TALON® is a simple four-stage procedure that can be performed in 10 min using a microcentrifuge: 1) After placing the column in a 2 ml microcentrifuge tube, equilibrate by adding binding buffer and centrifuge. 2) Add sample, centrifuge. 3) Wash with binding buffer, centrifuge. 4) Elute the target protein with elution by centrifugation.

 

Sample and buffer preparation
Refer to Purification using TALON® Superflow  for a general procedure for sample and buffer preparation.


Purification

Run purifications on His SpinTrap™ TALON® using a standard microcentrifuge. Place the column in a 2 ml microcentrifuge tube to collect the liquid during centrifugation. Use a new 2 ml tube for every step.


Remove storage solution

  1. Invert and shake the column repeatedly to resuspend the medium.
  2. Loosen the top cap one-quarter of a turn and break off the bottom closure.
  3. Place the column in a 2 ml microcentrifuge tube and centrifuge for 30 s at 70 to 100 × g.

Column equilibration

  1. Add 600 µl of binding buffer.
  2. Centrifuge for 30 s at 70 to 100 × g.

Sample application

  1. Add up to 600 µl of sample in one application.

After thorough cell disruption, it is possible to apply the unclarified sample directly without clogging. However, it is recommended to clarify the sample for optimal recovery.

  1. Seal the column with the top cap and bottom closure, and incubate the sample for 5 min with slow end-over-end mixing.
  2. Remove the top cap and bottom closure, and centrifuge for 30 s at 70 to 100 × g.

Several sample applications can be performed as long as the capacity of the column is not exceeded.

Wash

  1. Add 600 µl of wash buffer.
  2. To increase the yield, close the column with the top cap and bottom closure, and resuspend.
  3. Remove the top cap and bottom closure, and centrifuge for 30 s at 70 to 100 × g.
  4. Repeat this step once.

Elution

  1. Add 200 µl of elution buffer.
  2. To increase the yield, close the column with the top cap and bottom closure, and resuspend.
  3. Remove the top cap and bottom closure, centrifuge for 30 s at 70 to 100 × g, and collect the purified sample.
  4. Repeat this step once.

 

The first eluted 200 µl will contain the majority of the target protein.

Materials

     
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