Performing a Purification of IgG Antibodies with Protein G GraviTrap™

Protein G GraviTrap™ are gravity-flow columns prepacked with 1 mL of Protein G Sepharose^® 4 Fast Flow. The columns are designed for fast and efficient manual purification of monoclonal and polyclonal antibodies and antibody fragments from cell culture supernatant and biological fluids. The antibodies are simply captured with high specificity on protein G ligands in the gravity-flow columns. You do not need any other instrument with this protocol because the entire process relies on the flow of gravity. Together with the packaging that can be converted into a column stand (Workmate), parallel purification is made simple (Figure 3.4). The columns are reusable up to five times.

rProtein A Sepharose^® and a mix of Protein G Sepharose^® 4 Fast Flow and rProtein A Separose^® 4 Fast Flow are also available in GraviTrap™ format. The simple four-step procedure for purification is shown in Figure 3.5.

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 (28903059).

Purification

1. Cut off the bottom tip and remove the top cap. Pour off the column storage solution and place the column in the Workmate column stand. If needed, mount LabMate (PD-10 Buffer Reservoir) on top of the column.
2. Equilibrate the column with 10 mL of binding buffer.
3. After equilibration, add the sample. A volume of 1 to 20 mL is recommended. If the sample volume is less than 1 mL, dilute to 1 mL with binding buffer.
4. Add 15 mL binding buffer.
5. Add 3 to 5 mL of elution buffer. Collect the elution fraction. The collected elution fraction contains the purified protein.

As a safety measure to preserve the activity of acid-labile IgG, addition of 1 M Tris-HCl, pH 9.0 to the tubes used for collecting antibody-containing fractions (60 to 200 µl/mL eluted fraction) is recommended. In this way, the final pH of the sample will be approximately neutral.

The eluted fractions can be buffer exchanged using PD-10 Desalting columns or HiTrap Desalting columns (see Desalting and buffer exchange).
  6. After elution, regenerate the column by washing it with 5 to 10 mL of binding buffer. The column is now ready for a new purification.

Depending on the nature of the sample, Protein G GraviTrap™ columns may be reused up to five times consecutively. Reuse of the columns should only be considered when processing identical samples to avoid cross-contamination.