Protein G is a cell wall protein from group G streptococcal bacteria with a high affinity for the fragment crystallizable (Fc) region of monoclonal and polyclonal IgG-type antibodies from several different species, e.g., mouse, rat, human, etc. Capturem Protein G products use a recombinant Protein G engineered to remove the albumin binding domain as well as the cell wall and cell membrane-binding domains in order to improve specificity towards the Fc region of antibodies.

Capturem membranes have been engineered to have a very large surface area with low nonspecific background binding. The large surface area results in a high binding capacity (approximately 75 mg/ml of bed volume), about threefold higher than similar resins. In addition to providing a high binding capacity, the 3D structure of the membrane causes the loaded sample to flow through narrow pores with very short diffusion distances. This allows the membranes to rapidly bind to the target protein, yielding purified product in 5 minutes using Capturem miniprep columns and 15 minutes using Capturem maxiprep columns and high-throughput plate formats.

Comparison of Capturem Protein G and Capturem Protein A kits

The membranes in our Capturem Protein G minipreps, maxipreps, 24-well plates, and 96-well plates function in the same way as our Capturem Protein A membranes, except that they use Protein G to bind antibodies. Protein G has a slightly different affinity for antibodies than Protein A, and depending on the particular antibody and source species, Protein G might bind at a higher capacity than Protein A or vice versa. Typical binding capacities of Capturem Protein G Miniprep columns for serum from various source species are shown in Table I. For human IgGs, we expect that Protein G will be better for some isotypes. Even within a given isotype, there can be significant variability between IgG subclasses (Table II).

Binding capacity of Capturem Protein G Maxiprep columns

We used Capturem Protein G Maxiprep columns to purify mouse antibody produced from a hybridoma in a bioreactor (Figure 1). This test demonstrated the high binding capacity of the Protein G membrane when using optimized conditions. Typical antibody purifications yielded 1–2 mg per column (Table III).

Comparison of Capturem Protein G 96-well plates with resin spin plates

We tested the binding capacity of a Capturem Protein G 96-well plate side-by-side with a commercially available 96-well Protein G resin spin plate to purify dilute amounts of antibody from hybridoma supernatants (Figure 2). Antibodies were eluted from the Capturem membrane in a smaller volume using a shorter workflow (15 minutes instead of 1 hour), yielding a more concentrated antibody in less time.

Capturem Protein G 96-well plates have low variability for high-throughput applications

In addition to high-throughput screening, Capturem Protein G 96-well plates can also be used for high-throughput affinity purification upstream of applications such as mass spectrometry-based antibody quantification. We loaded eight replicates of two different hybridoma clone supernatants and a serum sample into individual plate wells to demonstrate the reproducibility and capacity of the Capturem Protein G 96-well plate (Figure 3).

Capturem Protein G products enable rapid IgG purification across a range of isotypes with a rapid workflow. Capturem Protein G 96-well plates offer consistent results for high-throughput applications, including affinity purification upstream of mass spectrometry-based antibody analysis, while Capturem Protein G Maxiprep columns can be used for projects requiring larger amounts of IgG. Capturem Protein G kits are appropriate for use with antibodies expressed in mammalian cells and whole serum, are compatible with various lysis buffers, and are available in miniprep, maxiprep, 24-well plate, and 96-well formats.

Capturem Protein G Miniprep columns were used according to the Capturem Protein G Miniprep protocol. First, the columns were equilibrated with 300 µl of Pierce Protein G Binding Buffer and then centrifuged at 1,000g for 1 min. Serum samples from sheep, goat, rat, mouse, human, rabbit, and horse (250 µl each) were diluted in 1 ml of Protein G binding buffer, and 600 µl of sample was loaded on an equilibrated column, followed by centrifugation at 1,000g for 1 min. The loading process was then repeated with another 600 µl of sample. The columns were then washed with 800 µl of Protein G binding buffer at 1,000g for 1 min. The bound antibody was then eluted with 300 µl of elution buffer (0.1 M glycine, pH 2.5) into a tube containing 30 µl of neutralization buffer (1 M Tris, pH 8.5) to neutralize the eluted antibody.

Capturem Protein G Maxiprep columns were used according to the Capturem Protein G Maxiprep protocol. First, the columns were equilibrated by loading 6 ml of Pierce Protein G binding buffer and centrifuging at 2,000g for 3 min. Next, 6.3-ml samples of mouse hybridoma supernatant from a bioreactor were diluted to 25 ml in Protein G binding buffer and then loaded onto the equilibrated columns by centrifugation at 2,000g for 3 min. We collected samples of the flowthrough for SDS-PAGE (Lane FT1 in Figure 1), then centrifuged the 25-ml samples through the columns a second time and collected another set of samples for SDS-PAGE (Lane FT2 in Figure 1). The columns were then washed once with 20 ml of Protein G binding buffer at 2,000g for 3 min. Lastly, four consecutive elutions were performed, each with 1 ml of elution buffer. The eluted fractions were analyzed using SDS-PAGE and quantified by measuring the absorbance at 280 nm with a NanoDrop 2000 spectrophotometer.

For the comparison of the Capturem 96-well plate and the Protein G resin 96-well plate, hybridoma supernatants from each clone were loaded onto each plate following the protocol for each product. The plates were first equilibrated, and then the supernatants were loaded and washed according to the protocols. Finally, the monoclonal antibody clones were eluted with 600 µl of elution buffer plus 60 µl of neutralization buffer in a collection tube.

To test the reproducibility of our 96-well plates, we loaded eight identical samples of two different hybridoma supernatants and animal sera into individual wells. Again, we equilibrated, loaded, washed, and eluted according to the protocol using Pierce Protein G Binding Buffer for all binding and washing steps. Eluates from various samples were quantified by measuring the absorbance at 280 nm with a NanoDrop 2000 spectrophotometer.