High-throughput Screening using GST MultiTrap™ 4B 96-well filter plates

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

GST MultiTrap™ 4B (Fig 5.4) is a prepacked, disposable 96-well filter plates for reproducible, high-throughput screening of GST-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. These filter plates simplify the purification screening and enrichment of up to 0.5 mg of GST-tagged proteins/well. After thorough cell disruption, it is possible to apply up to 600 µl of unclarified lysate directly to the wells in the 96-well filter plate without precentrifugation and/or filtration of the sample. It is recommended to extend the duration of mechanical/chemical lysis if the sample is too viscous after lysis; alternatively, include nucleases to disrupt nucleic acids. The GST-tagged proteins are eluted under mild, nondenaturing conditions that preserve protein structure and function.

The plates are packed with the AC media Glutathione Sepharose® 4 Fast Flow (4% highly cross-linked agarose beads) and Glutathione Sepharose® 4B (4% agarose beads), respectively. Each well contains 500 µl of a 10% slurry of Glutathione Sepharose® 4 Fast Flow or Glutathione Sepharose® 4B in storage solution (50 µl of medium in 20% ethanol). Note that binding depends on flow and may vary between proteins. Incubation of the sample with medium is needed, and optimization for ideal binding of the GST-tagged protein is recommended.

The 96-well filter plates with 800 µl wells are made of polypropylene and polyethylene. Characteristics of GST MultiTrap™ 4B are listed in Appendix 2 (Characteristics of Glutathione Sepharose® products).

Prepacked GST MultiTrap™ 4B plates give high consistency in reproducibility well-to-well and plate-to-plate. The repeatability of yield and purity of eluted protein is high. Automated robotic systems as well as manual handling using centrifugation or vacuum pressure can be used. The purification protocol can easily be scaled up because Glutathione Sepharose® is available in larger prepacked formats: GST GraviTrap, GSTrap™ FF, and GSTrap™ 4B (1 ml and 5 ml columns) and GSTPrep FF 16/10 (20 ml column).

GST MultiTrap 4B 96-well plates

Fig 5.4. GST MultiTrap™ 4B 96-well filter plates.

 

Sample preparation

Adjust the sample to the composition and pH of the binding buffer by additions from concentrated stock solutions; by diluting the sample with binding buffer; or by buffer exchange.

After thorough cell disruption, it is possible to apply unclarified lysate directly to the wells without precentrifugation and/or filtration of the sample. Apply the unclarified lysate to the wells directly after preparation, as the lysate may precipitate unless used immediately or frozen before use. New lysing of the sample can then prevent clogging of the wells when loading the plate.

Lysis with commercial kits could give large cell debris particles that may interfere with drainage of the wells during purification. This problem can be solved by centrifugation or filtration of the sample before adding it to the wells.

Buffer preparation

Use high-purity water and chemicals, and pass all buffers through a 0.45 µm filter before use.

Binding buffer:

Elution buffer:
PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), pH 7.3

50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

1 to 20 mM DTT can be included in the binding and elution buffers to reduce the risk of oxidation of free -SH groups on GST, which may cause aggregation of the tagged target protein, resulting in lower yield of GST-tagged protein.

 

Centrifugation procedure for high-throughput screening

Preparing the filter plate

  1. Peel off the bottom seal from the 96-well filter plate. Be sure to hold the filter plate over a sink to accommodate any leakage of storage solution when removing the bottom seal.
  2. Hold the filter plate upside down and gently shake it to dislodge any medium adhering to the top seal. Return the filter plate to an upright position.
  3. Place the filter plate against the bench surface and peel off the top seal.

Note: If the medium has dried out in one or several wells, add buffer to rehydrate it.
The performance of the medium is not affected.

  1. Position the filter plate on top of a collection plate.

Note: Remember to change or empty the collection plate as necessary during the following steps.

  1. Centrifuge the filter plates for 2 min at 500 × g, to remove the storage solution from the medium.
  2. Add 500 µl of deionized water/well. Centrifuge for 2 min at 500 × g.
  3. Add 500 µl of binding buffer/well, and mix briefly to equilibrate the medium. Centrifuge for 2 min at 500 × g. Repeat once.


Centrifugation procedure

Do not apply more than 700 × g during centrifugation.

  1. Apply unclarified or clarified lysate (maximum 600 µl per well) to the wells of the filter plate and incubate for 3 min.

Note: If the yield of protein is too low, increase the incubation time and/or gently agitate the filter plate to effect mixing.

  1. Centrifuge the plate at 100 × g for 4 min or until all the wells are empty. Discard the flowthrough.
  2. Add 500 µl of binding buffer per well to wash out any unbound sample. Centrifuge at 500 × g for 2 min. Repeat once or until all unbound sample is removed.

Note: Removal of unbound material can be monitored as A280. For high purity, A280 should be < 0.1.

  1. Add 200 µl of elution buffer per well and mix for 1 min.

Note: The volume of elution buffer can be varied (50 µl to 100 µl per well), depending on the concentration of target protein required. Volumes in the collection plate that are too small may give inaccurate absorbance values.

  1. Change the collection plate and centrifuge at 500 × g for 2 min to collect the eluted protein. Repeat twice or until all of the target protein has been eluted.

Note: The yield can usually be monitored for each fraction as A280 reading. If uncertain of required volume, change the collection plate between each elution to prevent unnecessary dilution of the target protein.

If the yield of eluted target protein is low, the incubation time can be increased.
 

Vacuum procedure for high-throughput screening

If problems with foaming, reproducibility, or bubbles in the collection plate occur using vacuum, the centrifugation procedure should be considered. The distance between the filter plate and the collection plate is critical and should be kept small (~5 mm); adjust the distance if necessary.

  1. Peel off the bottom seal from the 96-well filter plate. Be sure to hold the filter plate over a sink to accommodate any leakage of storage solution when removing the bottom seal.
  2. Hold the filter plate upside down and gently shake it to dislodge any medium adhering to the top seal. Return the filter plate to an upright position.
  3. Place the filter plate against the bench surface and peel off the top seal.

Note: If the medium has dried out in one or several wells, add buffer to rehydrate it. The performance of the medium is not affected.

  1. Position the filter plate on top of a collection plate.

Note: Remember to change or empty the collection plate as necessary during the following steps.

  1. Set the vacuum to -0.15 bar. Place the filter plate and collection plate on the vacuum manifold to remove the ethanol storage solution from the medium.
  2. Add 500 µl of deionized water to each well. Apply a vacuum to remove the water from the wells.
  3. Add 500 µl of binding buffer to each well to equilibrate the medium. Apply a vacuum as in step 5. Repeat this step once. The filter plate is now ready for use.

 

Preparing the filter plate

Vacuum procedure

If a robotic system is used for purification, the vacuum must be adjusted according to methods applicable to the system.

Do not apply a pressure in excess of -0.5 bar during vacuum operation.

  1. Apply unclarified or clarified lysate (maximum 600 µl per well) to the wells of the filter plate and incubate for 3 min.

Note: If the yield of protein is too low, increase the incubation time and/or gently agitate the filter plate.

  1. Apply a vacuum of -0.15 bar until all the wells are empty. Slowly increase the vacuum to -0.30 bar and turn off the vacuum after approximately 5 s. Discard the flowthrough.

Increasing the vacuum too quickly can result in foaming under the filter plate and subsequent cross-contamination of samples.

  1. Add 500 µl of binding buffer per well to wash out any unbound sample. Remove the solution as in step 2. Repeat once or until all unbound sample is removed.

Note: Removal of unbound material can be monitored as A280. For high purity, A280 should be < 0.1.

  1. Add 200 µl of elution buffer per well and mix for 1 min.

Note: The volume of elution buffer can be varied (50 µl to 100 µl per well), depending on the concentration of target protein required.

  1. Change the collection plate and apply vacuum as in step 2 to collect the eluted protein. Repeat twice or until all of the target protein has been eluted.

Note: The yield can usually be monitored for each fraction as A280 reading. If uncertain of required volume, change the collection plate between each elution to prevent unnecessary dilution of the target protein.

If the yield of eluted target protein is low, the incubation time can be increased.

Materials

     
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