Using Antibody-Binding Proteins for Antibody Purification
Choose a format based on the specific application and scale
by Alice Alegria-Schaffer, M.S. - 02/24/12
Protein A, Protein G, Protein A/G and Protein L are antibody-binding proteins useful for a variety of applications but are typically used while immobilized onto a solid support. Once immobilized, antibody-binding proteins are effective for antibody purification or immunoprecipitation of a target antigen. The particular species and class of antibody to be purified determines which immobilized protein is most appropriate (see Tech Tip #34 for specific binding information). Conveniently, these proteins are available already immobilized onto an agarose resin and in a variety of formats (Table 1). Which format to use depends on the specific application and the scale of the experiment.
Table 1. Formats available for antibody-binding proteins immobilized onto agarose resin†
|Antibody-binding Protein||Spin Column||Drip Column||Kit (spin/drip)||FPLC Cartridge||Bulk Resin||Spin Plate|
|Protein A||N/A||1mL||1mL drip||N/A||5mL, 25mL||N/A|
|Protein A High-Capacity||0.2mL, 1mL, 5mL||N/A||0.2mL, 1mL spin||1mL, 5mL||1mL, 5mL, 25mL||Yes|
|Protein G||0.2mL, 1mL, 5mL||N/A||0.2mL, 1mL spin||1mL, 5mL||2mL, 10mL, 25mL||Yes|
|Protein A/G||0.2mL, 1mL, 5mL||N/A||0.2mL, 1mL spin||1mL, 5mL||3mL, 15mL||N/A|
|Protein L||0.2mL, 1mL, 5mL||N/A||0.2mL, 1mL spin||1mL, 5mL||2mL, 10mL||N/A|
|†The formats listed in this table are part of Thermo Scientific Pierce Protein Biology Products. Some of these antibody-binding proteins are also available immobilized onto magnetic beads and other resin types. Other formats might be available from other suppliers.|
The Thermo Scientific NAb Spin Columns (Figure 1) are convenient for rapid, small-scale applications. The amount of IgG purified varies depending upon the sample type and the specific spin column used (Table 2). The spin-column format minimizes the time required to produce a purified antibody preparation.
Table 2. Choose a spin column (resin volume) based on sample size
|Sample Size (mL)||Resin (mL)||IgG Yield (mg)|
The antibody purification kits contain all the tubes and buffers required. The advantages of the kit format are convenience and consistency with regards to buffer formulations and resin that is pre-packed in the ready-to-use columns. Most of the kits contain spin columns; however, immobilized Protein A is still available in the drip format.
The Thermo Scientific Pierce Chromatography Cartridges (Figure 2) are pre-packed devices compatible with the major automated liquid-chromatography systems or for manual syringe processing. The cartridges attach directly to ÄKTA* or FPLC Systems without additional connectors. The included accessory pack enables the cartridges to be adapted for use with Luer-Lok* Syringe Fittings or 1/16” tubing. The cartridges provide fast, easy and reproducible chromatographic separations.
The bulk resin is available in various package sizes, including custom sizes to suit large-scale needs. The bulk resin is useful for assay development and protocol customization. The bulk resin is an economical way to optimize protocols, while providing the flexibility for assay size and format.
96-Well filter plates
The Thermo Scientific Protein A and Protein G Spin Plates for IgG Screening offer a high-throughput format for quick purification and screening of antibodies and for immunoprecipitation (IP). Each well of the 96-well spin plate can process 10-100mL of serum, cell culture supernatant or ascites samples.
We evaluated antibody-binding resins in different formats. Each format enabled effective antibody purification (Figures 3-5). Because the antibody-binding proteins have different affinities, depending on the type and source of IgG, results can vary.
The antibody-binding proteins enable effective antibody purification in all formats. Which format to use depends on the specific application and the scale of the experiment. For low amounts of antibody in 0.25-2mL (1-14mg per purification), spin columns provide a fast method; the pre-packed drip columns can process larger volumes and purify up to 175mg of antibody. Drip columns, however, require longer incubation and manual sample collection. Pre-packed FPLC chromatography cartridges provide a similar level of purification to drip-columns in an automated format. For screening small amounts of antibody (< 1mg antibody per sample) in a high-throughput format, the 96-well spin plates are optimal.
Thermo Scientific Pierce Protein A Plus Agarose is a versatile, high-performance affinity resin for antibody purification that is available as bottled agarose beads, pre-packed spin columns, 96-well filter plates and complete IgG purification kits.
Pierce Protein A Plus Agarose consists of purified native Protein A that has been covalently immobilized at high density onto high-quality crosslinked 6% beaded agarose (CL-6B). Among the many available varieties of immobilized Protein A affinity resins, this one provides the most versatile combination of chromatographic features for high yield and high purity purification of whole IgG from mammalian serum samples. The agarose beads have physical and chemical properties suitable for many affinity purification systems.
Features of Pierce Protein A Plus Agarose:
- Native Protein A – immobilized Protein A is ideal for polyclonal IgG purification from human, rabbit, pig, dog or cat serum
- Agarose resin – support is crosslinked 6% beaded agarose (CL-6B), the most popular resin for protein affinity purification methods
- Inert and stable – superior manufacturing method immobilizes Protein A by charge-free, leach-resistant covalent bonds, resulting in low nonspecific binding and enabling multiple uses without decline in yield
- High capacity – this “Plus” variety of Pierce Protein A Agarose has a dense load of immobilized Protein A, providing a binding capacity greater than 34 mg human IgG/mL resin (approx. 16 to 17 mg mouse IgG/mL resin)
Learn more about Thermo Scientific Pierce Protein A Plus Agarose