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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. | ||||||
Spin columns
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.
Figure 1. Thermo Scientific NAb Spin Columns are available in three sizes. The 0.8mL, 2mL and 10mL centrifuge columns are filled respectively with 0.2mL, 1mL and 5mL of Protein A, G, A/G or L beaded agarose resin. The columns are designed to fit microcentrifuge tubes, 15mL conical tubes and 50mL conical tubes, respectively. Column products containing resin refer to the filled bed volume in their names.
Table 2. Choose a spin column (resin volume) based on sample size
| Sample Size (mL) | Resin (mL) | IgG Yield (mg) |
|---|---|---|
| 0.025-0.5 | 0.2 | 0.1-1 |
| 0.5-2 | 1 | 1-13 |
| 2-10 | 5 | 5-65 |
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.
Chromatography cartridges
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.
Figure 2. Schematic of the Thermo Scientific Pierce FPLC Cartridges.
Bulk resin
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.
Results and Discussion
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.
Figure 3. Spin columns provide easy antibody purification in less than 30 minutes. Sera (approximately 60mg protein) from different hosts were applied to the NAb Protein A, Protein G, Protein A/G and Protein L Spin Columns (1mL) and processed by the drip or spin method. For the spin method, samples were incubated with the resin for 10 minutes at room temperature by gentle end-over-end mixing. The resins were washed with 6mL of wash buffer and IgG eluted with 3mL (3 x 1mL) of elution buffer. Total time for the spin procedure was 19 minutes. For the drip (gravity flow) method, the samples were allowed to flow slowly through the column. The columns were washed with 15mL of wash buffer. The IgG was eluted with 5mL (5 x 1mL) of elution buffer. Total time for the drip procedure was 120 minutes. Filled and open triangles mark heavy and light chains (50 and 25kDa) of the purified IgG, the result of reducing SDS-PAGE. The strong band at 66kDa in the serum (S) lanes is albumin.
Figure 4. Chromatography cartridges provide high antibody yield and purity of human IgG. Normal human serum (60mg) in PBS was applied to a 1mL Thermo Scientific Pierce Protein A Cartridge and eluted with 0.1M glycine, pH 2.8, using a flow rate of 1mL/minute. The arrow denotes the start of the low pH elution. The yield of human IgG was 6.85mg. Fractions were separated by SDS-PAGE and the gel was stained with Thermo Scientific Imperial Protein Stain (Part No. 24615). M = MW marker, S = serum load, F = flow-through and E = elution.
Figure 5. The Protein A spin plate effectively purifies human IgG. Human serum (40µL) was purified using one well of the Pierce Protein A Spin Plate according to the product instructions. The collected samples and controls were prepared in reducing sample buffer, separated by Tris-glycine SDS-PAGE and stained with Imperial Protein Stain (Part No. 24615). M = MW marker, Ig = pure human IgG control, S = human serum, F = flow-through, E = elution.
Conclusions
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
For Research Use Only. Not for use in diagnostic procedures.