Perform IP and Co-IP Experiments with High Yield and Low Background

Protein A/G magnetic beads enable manual and automated applications

by Thermo Scientific Pierce Protein Biology Team - 06/01/11

The Thermo Scientific Pierce Protein A/G Magnetic Beads enable efficient immunoprecipitation (IP), co-immunoprecipitation (co-IP) and antibody purification with high purity and low background. These unique 1µm particles consist of a polymeric core coated with two layers of magnetite and sealed with a blocking agent to reduce nonspecific binding (Table 1; Figure 1). The bead surface is coupled with recombinant Protein A/G, which combines the IgG-binding domains of both Protein A and Protein G. Protein A/G enables capture of immunoglobulins from a wider range of species and antibody isotypes than either Protein A or Protein G alone. The beads can be used both manually with a magnetic stand and with automated platforms such as the Thermo Scientific KingFisher Instruments.

Figure 1. Diagram of Thermo Scientific Pierce Protein A/G Magnetic Beads.

The Pierce Protein A/G Magnetic Beads can be used to purify antibodies from biological fluids such as serum. When compared with Protein A and Protein G magnetic beads from other suppliers, Pierce A/G Magnetic Beads purify significantly more antibody from rabbit (Figure 2A) and mouse (Figure 2B) serum with less background. Additionally, the Pierce A/G Magnetic Beads have a four-fold higher binding capacity (per milligram of beads) for purified rabbit IgG than magnetic beads from another supplier (Figure 3).

Figure 2. Isolate more IgG from rabbit and mouse serum with less background. Using a KingFisher Flex with a 96 deep well plate, IgG was purified from rabbit (Panel A) and mouse (Panel B) serum (5mg total protein) using 50 μL of Pierce Protein A/G Magnetic Beads and Protein A and Protein G magnetic beads from other suppliers. The beads were washed with Tris-buffered saline containing 0.05% Tween 20 Detergent (TBST), incubated 1 hour with serum diluted in TBST, washed three times, and eluted with 0.1M glycine, pH 2.8 for 10 minutes at room temperature. The eluates were resolved by SDS-PAGE and stained with Thermo Scientific Imperial Protein Stain (Cat. No. 24615). The IgG heavy chain bands were quantified by densitometry. The values for each set of duplicate bands were averaged and expressed as a percentage of the average for the Pierce Protein A/G Beads.

Figure 3. The rabbit IgG binding capacity of Thermo Scientific Pierce Protein A/G Magnetic Beads is approximately four-fold higher than magnetic beads from a leading supplier. Pierce Protein A/G Magnetic Beads and Protein A and Protein G magnetic beads from another supplier were added to a 96 deep-well plate (1mg beads per well). Using the KingFisher 96 Instrument, the beads were washed with phosphate-buffered saline containing 0.05% Tween 20 Detergent (PBST) and incubated for 1 hour with varying amounts of purified rabbit IgG (20-200 μg). After binding, the beads were washed three times with PBST and eluted at 96°C with SDS-PAGE reducing sample buffer. Binding was calculated by subtracting the amount of protein in the flow-through from the amount loaded using the Thermo Scientific Pierce BCA Protein Assay (Cat. No. 23225).

The Pierce Classic Magnetic IP/Co-IP Kit enables efficient immunoprecipitation of a target protein from cell lysates (Figure 4). This kit can also effectively isolate intact protein complexes and co-IP proteins that are bound to the target protein. For example, IP of Cdk1 will co-IP cyclin B if the cells are synchronized in late G2 phase. The Pierce Protein A/G Magnetic Beads co-immunoprecipitated cyclin B from U2OS cell lysate with equal or higher yield than Protein A and Protein G beads from other suppliers (Figure 5A). Silver stain of the bead eluates revealed negligible background (excluding antibody) with all beads tested (Figure 5B).

Figure 4. The Thermo Scientific Pierce Classic Magnetic IP/Co-IP Kit effectively immunoprecipitates Cdk1. U2OS (human osteosarcoma) cells were synchronized in late G2 phase by serum starvation followed by growth in 20% fetal bovine serum for 18 hours before harvest. Cells were lysed in IP Lysis/ Wash Buffer, and 0.75 mg of lysate (per sample) was incubated with and without anti-Cdk1 antibody overnight at 4°C. The Pierce Protein A/G Magnetic Beads and Protein A and Protein G magnetic beads from three suppliers were added (50 μL each) to a 96 deep-well plate. Using the KingFisher Flex Instrument, beads were washed with IP Lysis/Wash Buffer, incubated for 1 hour with the antigen sample/antibody mixture, washed twice with IP Lysis/Wash Buffer containing 0.5 M NaCl, washed once with water, and eluted with SDS-PAGE reducing sample buffer for 10 minutes at room temperature. The eluates were resolved by SDS-PAGE and analyzed by western blot for Cdk1. The Pierce Protein A/G Magnetic Beads were able to effectively IP Cdk1 with a higher yield than beads from other suppliers.

Figure 5. Thermo Scientific Pierce Classic Magnetic IP/Co-IP Kit effectively co-immunoprecipitates cyclin B and Cdk1. U2OS cells were synchronized and samples were prepared as described in Figure 3. Eluates were resolved by SDS-PAGE and analyzed by western blot (Panel A) for cyclin B or silver-stained (Panel B). The Pierce Protein A/G Beads were able to effectively co-IP cyclin B with yields higher than or equal to beads from other suppliers. The Pierce Beads bound the same or more antibody than Protein A and Protein G magnetic beads from other suppliers (Panel B). All beads had negligible nonspecific binding.