Purification of Histidine-Tagged Proteins using His MultiTrap™ TALON®

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

His MultiTrap™ TALON® 96-well filter plates are prepacked with 50 µl of TALON® Superflow per well. Each plate provides highly reproducible high-throughput screening and rapid small-scale purification of histidine-tagged proteins. Typical applications include expression screening of different constructs, screening for solubility of proteins, and optimization of the conditions for small-scale parallel purification. Purification of up to 1 mg of histidine-tagged proteins per well is possible. The 96-well plate format gives great flexibility, both when working with automated robotic systems and when manually using centrifugation or vacuum. Consistent well-to-well and plate-to-plate performance ensures high reproducibility in yield and purity. See Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose® excel, TALON® Superflow, and uncharged IMAC Sepharose® products) for the main characteristics of His MultiTrap™ TALON.

His MultiTrap TALON

Fig 3.38. His MultiTrap™ TALON® prepacked 96-well plates for expression screening and small-scale parallel purification of histidine-tagged proteins.

 

Sample and buffer preparation

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

 

Purification

Purification with His MultiTrap™ TALON® can be performed either by using centrifugation or vacuum pressure.


Purification protocol using centrifugation

This protocol is a general guideline for the purification with His MultiTrap™ TALON. Optimization may be required depending on source and type of protein.

Remove storage solution

  1. Remove the bottom seal.
  2. Gently shake the 96-well filter plate while holding it upside down, to remove any medium stuck on the top seal. Place the plate in upright position.
  3. Remove the top seal from the plate while holding it against the bench surface.
  4. Position the plate on top of a collection plate.

Remember to change or empty the collection plate when necessary during the following steps.

  1. Centrifuge the plates for 2 min at 500 × g, to remove the storage solution from the medium.


Prewash

  1. Add 500 µl of deionized water per well.
  2. Centrifuge for 2 min at 500 × g.


Equilibrate

  1. Add 500 µl of binding buffer/well and mix briefly, to equilibrate the medium.
  2. Centrifuge for 2 min at 500 × g.
  3. Repeat once.


Load sample

  1. Apply sample to the wells (maximum 600 µl/well).

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

  1. Incubate for 3 min. (Increase the incubation time if the yield is too low.)
  2. Remove the flowthrough by centrifuging for 4 min at 100 × g (or until all wells are empty).


Wash

  1. Add 500 µl of wash buffer/well to wash out unbound sample.
  2. Centrifuge for 2 min at 500 × g.
  3. Repeat once (or until all unbound sample is removed).


Elute

  1. Add 200 µl1 of elution buffer/well and mix for 1 min.
  2. Change collection plate and centrifuge the plates for 2 min at 500 × g, and collect the fractions.
  3. Repeat twice (or until all target protein has been eluted).
  4. If required, change collection plate between each elution (to prevent unnecessary dilution of the target protein).

1 The volumes can be varied depending on which concentration of target protein is needed (e.g., 50 or 100 µl of elution buffer/well).


Purification protocol using vacuum pressure

This protocol is only a general guideline for the purification with His MultiTrap™ TALON. Optimization may be required depending on source and type of protein.

If a problem with cross-contamination due to foaming, poor reproducibility or bubbles in the collection plate occurs using vacuum, decrease load of protein (< 0.5 mg protein bound to medium). If this does not solve the problem, the centrifugation protocol should be considered.

To avoid cross-contamination, the distance between MultiTrap™ and collection plate should be as narrow as possible (not more than 5 mm).

Use deep round well collection plates (500 µl) to avoid splashes between wells.

A vacuum pressure of -150 mbar (30 s) followed by -300 mbar (<3 s) should be used during elution of purified protein.

Vacuum parameters need to be optimized for each vacuum manifold.

 

Purification

Remove storage solution

  1. Remove the bottom seal.
  2. Gently shake the 96-well filter plate while holding it upside down, to remove any medium stuck on the top seal. Place the plate in an upright position.
  3. Remove the top seal from the plate while holding it against the bench surface.
  4. Place the 96-well filter plate on the vacuum manifold. Remove the storage solution from the medium by applying a vacuum pressure of -300 mbar for 20 s.

Position the filter plate on top of a collection plate. Remember to change or empty the collection plate when necessary during the following steps.


Prewash

  1. Add 500 µl of deionized water/well.
  2. Remove the water from the wells by applying a vacuum pressure of -300 mbar for 20 s.


Equilibrate

  1. Add 500 µl of binding buffer/well to equilibrate the medium.
  2. Remove the solution by applying a vacuum pressure of -300 mbar for 20 s.
  3. Repeat once.


Load sample

  1. Apply sample to the wells (maximum 600 µl/well).

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

  1. Incubate for 3 min. (Increase the incubation time and gently mix the filter plate if the yield is too low.)

In purifications using a robot, the vacuum must be adjusted to methods applicable to the robot.

  1. Remove the flowthrough by applying a vacuum pressure of -150 mbar until all wells are empty (30 to 50 s).


Wash

  1. Add 500 µl of wash buffer/well to wash out unbound sample.
  2. Remove the solution by applying a vacuum pressure of -150 mbar for 30 s.

-300 mbar for 20 s could also be used if the waste will not be collected.

  1. Repeat twice (or until all unbound sample is removed).


Elute

  1. Add 200 µl of elution buffer1 and mix for 1 min.
  2. Change/add collection plate and elute the sample using vacuum by applying a vacuum pressure of -150 mbar for 30 s followed by applying a vacuum pressure of -300 mbar for < 3 s, or until all droplets under the plate are removed.
  3. Repeat twice (or until all target protein has been eluted).
  4. If required, change collection plate between each elution to prevent unnecessary dilution of the target protein.

1 The volumes can be varied depending on the concentration of target protein needed, e.g., 50 or 100 µl of elution buffer/well.

Increasing the vacuum too fast can give foam under the filter plate, and cross-contamination can occur.

Materials

     
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