<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Getting Started

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You have a protein that you want to purify out of a complicated mixture. This protein has a 6xHis purification tag, which chelates Ni ions. You can use a Ni-His resin to bind proteins with 6xHis tags. This resin is a suspension of microscopic gel beads with Ni ions covalently linked. Bound proteins can then be washed to remove non-tagged proteins, then eluted in a buffer with high concentration (~ 500 mM) Imidazole. Imidazole competes with 6xHis to bind the Ni ions, ultimately displacing your target proteins and releasing them from the affinity resin.

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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					 | $72.46 | 4C to 30C | [[link](<https://us.vwr.com/store/catalog/static_catalog.jsp?catalog_number=97064-756>)] |

| HEPES | HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle | Sigma-Aldrich | H3375-500G | $431 | 4C to 30C | [link] | | Imidazole | ReagentPlus® Imidazole, Crystalline, 99% | Sigma-Aldrich | I202-500G | $120 | 4C to 30C | [link] | | Ammonium Chloride | Ammonium chloride,ACS reagent, ≥99.5% | Sigma-Aldrich | 213330-500G | $73.50 | 4C to 30C | [link] | | Sodium Chloride | Sodium Chloride, Redi-Dri™, anhydrous, free-flowing, ACS, ≥99% | Sigma-Aldrich | 746398-1KG | $133.00 | 4C to 30C | [link] | | Potassium Chloride | Potassium Chloride, ACS reagent, 99.0-100.5% | Sigma-Aldrich | P3911-1KG | $146.00 | 4C to 30C | [link] | | Magnesium Chloride hexahydrate | BioXtra Magnesium chloride, Hexahydrate, Powder or Crystals, ≥99.0% | Sigma-Aldrich | M2670-100G | $50.50 | 4C to 30C | [link] | | TCEP-HCl (0.5M; presuspended) | Thermo Scientific, Bond-Breaker® Tris[2-Carboxyethyl]phosphine Neutral Solution (TCEP), Application=Water-Soluble Reducing Agent, Molecular Weight=286.65, Size=5 mL | Thermo Scientific | 77720 | $175.65 | 4C to 30C | [link] |

<aside> <img src="/icons/merge_gray.svg" alt="/icons/merge_gray.svg" width="40px" /> Prerequisite Protocols

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• [ ] Prepare P1, P2 and 2x Storage buffers: Make Protein Purification Buffers and Media.
• [ ] Assemble and equilibrate columns: Prepare Columns.
• [ ] Prepare clarified lysates: Lyse Bacteria by Sonication.

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol

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• [ ] Prepare the following buffers; chill at 4C:

  • [ ] 50 mL P1 buffer per sample.
  • [ ] 6 mL P2 buffer per sample.
  • [ ] 20 mL 4x P3 storage buffer per sample.

• [ ] Assemble one (1) gravity column (2 mL resin bed) per sample.

• [ ] Wash each column with 5 CV of Ultrapure water. For this protocol, we will assume you are using a 2 mL resin bed.

  • Notes: “CV” = Column Volume

• [ ] Equilibrate each column with ≥ 5 CV (10 mL) cold P1 buffer.

• [ ] Load up to 5 CV (10 mL) clarified lysate per column. (Optionally) capture flowthrough for later analysis (see Pierce660 Assay and Protein Gel).

  • Notes: if you have >10 mL lysate, load in tranches.

• [ ] Wash column with ≥ 5 CV (10 mL) cold P1 buffer. Allow buffer to flow through. (Optionally) capture flowthrough for later analysis; see Pierce660 Assay and Protein Gel.

• [ ] Elute sample with 3 CV (6 mL) cold P2 buffer. Capture flow through in 15 mL centrifuge tube.

<aside> <img src="/icons/book_gray.svg" alt="/icons/book_gray.svg" width="40px" /> Resources and References

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• Papers

  • Original PURE paper

  Cell-free translation reconstituted with purified components

  • OnePot PURE

  A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

  OnePot PURE Cell-Free System | Text Page

<aside> <img src="/icons/megaphone_gray.svg" alt="/icons/megaphone_gray.svg" width="40px" /> Credits

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Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)

• Developers
  • Murray Lab
  • Freemont Lab
  • Shimizu lab