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

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You want to purify proteins and you’re going to use gravity columns with affinity resin. This protocol will show you how to set up these columns.

We use Ni-His columns to purify PURE proteins. These columns are packed with microscopic gel beads containing covalently bound Ni in their interior. This resin specifically bind proteins with a poly Histidine tag (e.g., 6xHis). The His residues chelate the Ni and bind tightly. Proteins are then eluted with Imidazole, which competes with the His tags to chelate Ni.

<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] | | 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/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol

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• [ ] Prepare Stock Solutions and P1 Wash Buffer as specified in Make Protein Purification Buffers and Media

• [ ] Assemble columns.

  • [ ] Cut tips off of columns off using scissors or a razor blade.
  • [ ] Pack a filter into the bottom of the column (we use the back end of a cell spreader).
  • Notes: columns have ridges!

• [ ] Wash empty columns.

  • [ ] Clamp columns to a lab stand and put a large beaker (≥ 500 mL) underneath to capture flowthrough.
  • [ ] Wash columns and filters with 10 mL Ultrapure water.

• [ ] Load and Equilibrate Ni2+ affinity Resin into columns

  • [ ] Resuspend Ni2+ resin (50% v/v suspension) by shaking.
  • [ ] Add 2 mL resin by pipette to each column.
  • [ ] Equilibrate columns with 10 CV of cold P1 Wash Buffer (4C) and discard flowthrough.
  • Notes: “CV” = Column Volume

• [ ] Store Columns.

  • [ ] Seal each column with a cap and bung.
  • [ ] Store columns at 4C for up to 48 hrs.
  • Notes: store columns long term in EtOH (20%)

<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