Thermo Scientific Pierce Protein A/G磁気ビーズを用いて、低バックグラウンドな免疫沈降(IP)や共免疫沈降(co-IP)、ならびに高純度の抗体精製が行えます。独自の1 μm粒子は、2層の磁鉄鉱でコーティングされ、非特異的な結合を減らすためにブロッキング剤でシールした重合体のコアからなっています（表1、図1）。ビーズ表面は、Protein AとProtein G両方のIgG結合ドメインを融合した組換えProtein A/Gと結合しています。Protein A/Gは、Protein AまたはProtein G単独よりも、幅広い種および抗体アイソタイプから免疫グロブリンを捕捉することが可能です。これらのビーズは、磁気スタンドおよび自動プラットフォーム（Thermo Scientific KingFisherの機器など）で使用できます。

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

Pierce Protein A/G磁気ビーズは、血清などの生体液から抗体を精製するのに使用することができます。他社製のProtein AおよびProtein G磁気ビーズと比べて、Pierce A/G磁気ビーズを用いることにより、ウサギ血清（図2A）やマウス（図2B）血清から、より少ないバックグラウンドで非常に多くの抗体を精製できます。さらに、Pierce A/G磁気ビーズは、他社製の磁気ビーズよりも、精製ウサギIgGに対して4倍高い結合容量（ビーズ1 mgあたり）をもっています（図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).

Pierce Classic Magnetic IP/Co-IPキットは、細胞溶解液から標的タンパク質を効率的に免疫沈降させることが可能です（図4）。本キットは、標的タンパク質に結合したタンパク質複合体およびco-IPタンパク質を効果的に単離することも可能です。例えば、G2期後半にシンクロする細胞では、Cdk1のIPによりサイクリンBを免疫共沈降させることができます。Pierce Protein A/G磁気ビーズは、他社製のProtein AおよびProtein Gビーズと比べて同等以上の収量で、U2OS細胞ライセートからサイクリンBを免疫共沈降させることができます（図5A）。ビーズ溶出物の銀染色では、試験したすべてのビーズで、バックグラウンド（抗体を除く）はごくわずかでした（図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.