Performing a Purification of IgG Antibodies with Protein G HP MultiTrap™

Extracted from Affinity Chromatography Vol. 1: Antibodies, GE Healthcare, 2016
 

Protein G HP MultiTrap™ are 96-well plates prepacked with Protein G Sepharose® High Performance. Protein G HP MultiTrap™ is a versatile tool for screening of different proteins and for preparation of protein samples, enrichment of proteins of interest from clarified cell lysates and biological fluids, and small-scale purification of antibodies (Figure 3.6). Purification runs are performed in parallel, which ensures fast and reliable capture of antibodies from a large number of complex samples.

Each pack of Protein G HP MultiTrap™ contains four prepacked multiwell plates and protocols for protein enrichment of target antibody-antigen complexes by immunoprecipitation and for small-scale antibody purification. Collection plates for collecting eluted, purified antibody are available separately.

The procedure for small-scale screening and purification of antibodies is described below. For a description of the immunoprecipitation procedure, download Instructions 28906773.

Protein G HP MultiTrap™ 96-well plates

Figure 3.6. Protein G HP MultiTrap™ 96-well plates are used for rapid, parallel screening and small-scale purification of monoclonal and polyclonal antibodies at small scale.

 

Sample preparation

Refer to Desalting and buffer exchange for general considerations.

The sample should have a pH around 7.0 before applying to the column. If required, adjust sample conditions to the pH and ionic strength of the binding buffer by either buffer exchange on a desalting column (see Desalting and buffer exchange) or dilution and pH adjustment.

 

Buffer preparation

Binding buffer: 0.02 M sodium phosphate, pH 7.0

Elution buffer: 0.1 M glycine-HCl, pH 2.7

Neutralizing buffer: 1 M Tris-HCl, pH 9.0

Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.45 µm filter before use.

Buffers can be prepared from the 10× stock solutions of binding and elution buffers supplied with Ab Buffer Kit (GE28903059).

 

Purification

  1. Prepare two collection plates with 15 µl neutralizing buffer per well.

To preserve the activity of acid-labile IgG, we recommend adding 15 µl of 1 M Tris-HCl pH 9.0 to collection wells in the collection plate, which ensures that the final pH of the sample will be approximately neutral.

  1. Invert and gently shake the MultiTrap™ plate to resuspend the medium. Remove top and bottom seals and place the MultiTrap™ plate on a collection plate. Centrifuge for 1 min at 70 to 100 × g to remove the storage solution.
  2. Equilibrate by adding 300 µl binding buffer. Centrifuge for 30 s at 70 to 100 × g.
  3. Bind antibody by adding maximum 300 µl of the antibody sample. Incubate for 4 min while gently mixing. Centrifuge for 30 s at 70 to 100 × g.
  4. Wash by adding 300 µl binding buffer and centrifuge for 30 s at 70 to 100 × g. Repeat this step.
  5. Replace the collection plate with a fresh collection plate prepared in step 1.
  6. Add 200 µl of elution buffer to elute the antibody and centrifuge for 30 s at 70 × g. Collect the eluate. Repeat this step. Most of the bound antibody is eluted after two elution steps.

Desalt and/or transfer purified IgG fractions to a suitable buffer using a desalting column (see Desalting and buffer exchange).

 

Storage

Store in 20% ethanol at 2°C to 8°C.

Materials

     
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