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Agarose Co-IPおよびプルダウンキット
プルダウンは共免疫沈降（Co-IP）の発展型で、標的タンパク質（つまり抗原）を対応する抗体とともに捉えて単離します。タグベース IP およびプルダウンの手法は、ビオチン化された his-、GST-、c-Myc、HA タグを用いてタンパク質相互作用標的を捉えるため、抗体の代わりに bait タンパク質を使います。
- 豊富な選択肢—多様な標識 bait タンパク質や溶出条件に対応するため、またダウンストリーム解析のニーズに合わせるための様々な手法をご用意しています
- Complete—kits include all controls and lysis, binding, wash, and elution buffers
- Fast—spin columns and collection tubes are included that shorten the protocol and minimize sample handling
- Flexible—can scale up or down as needed using adaptable protocol
 |  |  |  |  | | |
|---|---|---|---|---|---|---|
| Pierce Biotinylated Protein Interaction Pull-Down Kit | EZ-Link Desthiobio-tinylation and Pull-Down Kit | Pierce c-Myc Tag IP/Co-IP Kit | Pierce HA Tag IP/Co-IP Kit | Pierce GST Protein Interaction Pull-Down Kit | Pierce His Protein Interaction Pull-Down Kit | |
| Target | Biotinylated proteins | Biotinylated proteins | c-Myc–tagged recombinant protein | HA-tagged recombinant protein | GST-tagged recombinant protein | His-tagged recombinant protein |
| Base bead | Streptavidin agarose resin, | High-capacity streptavidin agarose resin | Anti-c-Myc agarose resin | Anti-HA agarose | Glutathione agarose | Cobalt chelate agarose resin |
| Binding capacity | 1–3 mg/mL | >10 mg/mL | 102 nmol/mL | >60 nmol/mL | 8–10 mg/mL | 10–25 mg/mL |
| Non-specific binding | Low | Low | Lower | Lower | Lowest | Lowest |
| Elution conditions | Low pH (2.8) Buffer | Mild (free biotin buffer) | Low pH (2.0) buffer | Non-reducing sample buffer | 100 mM reduced glutathione | 250 mM imidazole solution |
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細菌ライセートからのIP中GST-HAおよびGST-c-Myc 溶出オプションの有効性
IPの結果は、Thermo Scientific Pierce HA IP/Co-IP Kitとc-Myc IP/Co-IP Kitで、適切なポジティブコントロールのライセートと、GST-HA および GST-cMyc それぞれに推奨された溶出オプションを用いて達成できます。溶出成分は各キットに含まれています。溶出1は溶出バッファで行い、溶出 2 は 2X 非還元のサンプルバッファで行いました。
| Lane # | A. GST-Tag Pull-Down | B. PolyHis-Tag Pull-Down |
|---|---|---|
| 1 | Lysate from E. coli expressing GST-tagged BIR2 (bait protein). | Lysate from E. coli expressing 9xHis-tagged wild-type Smac (bait protein). |
| 2 | Flow-through from the lysate in Lane 1 bound to an immobilized reduced glutathione support for 1 hour at 4°C. | Flow-through from the lysate in Lane 1 bound to an immobilized cobalt chelate support for 1 hour at 4°C. |
| 3 | Wash #1 of the support. | Wash #1 of the support. |
| 4 | Wash #2 of the support. (Washes 3-5 not shown.) | Wash #2 of the support. (Washes 3-5 not shown.) |
| 5 | Lysate from E. coli expressing 9xHis-tagged wild-type Smac (prey protein). | Lysate from E. coli expressing GST-tagged BIR2 (prey protein). |
| 6 | Flow-through from the lysate in Lane 5 is allowed to interact with the immobilized bait for 1 hour at 4°C. | Flow-through from the lysate in Lane 5 is allowed to interact with the immobilized bait for 1 hour at 4°C. |
| 7 | Wash #1 of the support. | Wash #1 of the support. |
| 8 | Wash #2 of the support. (Washes 3-5 not shown.) | Wash #2 of the support. (Washes 3-5 not shown.) |
| 9 | Bait control. Bait treated as described in Lanes 1-8 and subsequently eluted. No prey added – just binding buffer. | Bait control. Bait treated as described in Lanes 1-8 and subsequently eluted. No prey added – just binding buffer. |
| 10 | Prey control. Prey treated as described in Lanes 1-8 and subsequently eluted. No bait added – just binding buffer. | Prey control. Prey treated as described in Lanes 1-8 and subsequently eluted. No bait added – just binding buffer. |
| 11 | Elution of bait:prey complex (prepared in Lanes 1-8) from the support with 100 mM reduced glutathione. Western blotting confirms that the minor bands observed in Lanes 9 and 11 are degradation products of GST-tagged BIR2. | Elution of bait:prey complex (prepared in Lanes 1-8) from the support with 250 mM imidazole. |
For Research Use Only. Not for use in diagnostic procedures.