Overview¶
You have a protein that you want to purify out of a complicated mixture. This protein has a 6xHis purification tag, which chelates Ni²⁺ 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.
This protocol is derived from the original PURE system (Shimizu et al., 2001) and from the OnePot PURE method (Lavickova & Maerkl, 2019; Grasemann et al., 2021).
Important Information
Please read this section carefully. It contains important notes, resources, and safety information. Not all information included here is included in the lab-ready protocol.
Prerequisite Documentation
Materials and Equipment¶
Bill of Materials
| Name | Category | Product | Manufacturer | Part # | Price | Storage | Link |
|---|---|---|---|---|---|---|---|
| Columns | Columns | Empty Disposable PD-10 Columns | Cytiva | 17043501 | $258.30 | 4 °C to 30 °C | link |
| Ni-His Resin | Resin | NEBExpress Ni Resin | New England Biolabs | S1428S | $358.00 | 1 °C to 4 °C | link |
| 15 mL centrifuge tubes | Consumables | Corning® 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar™ Cap, Sterile, 50/Rack, 500/Case | Corning | 430790 | $230.44 | 4 °C to 30 °C | link |
| Potassium Hydroxide | Buffers | Potassium hydroxide - ACS reagent, ≥85%, pellets | Sigma-Aldrich | 221473-1KG | $99.90 | 4 °C to 30 °C | link |
| Hydrochloric acid (1M) | Buffers | Hydrochloric acid 1 N, Reagent Grade, Packaging=Amber Bottle, Size=500 mL | VWR | E447-500L | $72.46 | 4 °C to 30 °C | link |
| HEPES | Buffers | HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle | Sigma-Aldrich | H3375-500G | $431 | 4 °C to 30 °C | link |
| Imidazole | Buffers | ReagentPlus® Imidazole, Crystalline, 99% | Sigma-Aldrich | I202-500G | $120 | 4 °C to 30 °C | link |
| Ammonium Chloride | Buffers | Ammonium chloride, ACS reagent, ≥99.5% | Sigma-Aldrich | 213330-500G | $73.50 | 4 °C to 30 °C | link |
| Sodium Chloride | Buffers | Sodium Chloride, Redi-Dri™, anhydrous, free-flowing, ACS, ≥99% | Sigma-Aldrich | 746398-1KG | $133.00 | 4 °C to 30 °C | link |
| Potassium Chloride | Buffers | Potassium Chloride, ACS reagent, 99.0-100.5% | Sigma-Aldrich | P3911-1KG | $146.00 | 4 °C to 30 °C | link |
| Magnesium Chloride hexahydrate | Buffers | BioXtra Magnesium chloride, Hexahydrate, Powder or Crystals, ≥99.0% | Sigma-Aldrich | M2670-100G | $50.50 | 4 °C to 30 °C | link |
| TCEP-HCl (0.5M; presuspended) | Buffers | 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 | 4 °C to 30 °C | link |
Protocol¶
Prepare Columns¶
Prepare buffers: Prepare the following buffers; chill at 4 °C:
50 mL Wash Buffer per sample.
6 mL Elution Buffer per sample.
Assemble columns: Prepare one (1) gravity column per sample:
Cut the tips off the columns with scissors or a razor blade.
Pack a filter into the bottom of each column (we use the back end of a cell spreader).
Note: columns have ridges!
The filter needs to be pushed past two circular ridges on the inside of the column — one ~2 cm from the opening and another ~5 cm further down.
Wash empty columns: Clamp the columns to a lab stand with a large beaker (≥ 500 mL) underneath to capture flowthrough. Wash each column and filter with 10 mL Ultrapure water.
Load and equilibrate resin:
Resuspend the Ni²⁺ resin (50% v/v slurry) by shaking.
Add 2 mL resin slurry (≈ 1 mL settled bed) to each column by pipette.
Equilibrate each column with ≥ 10 CV (10 mL) cold Wash Buffer (4 °C); discard the flowthrough.
Note: “CV” = Column Volume
One “Column Volume” (CV) is the settled resin bed volume. Our 2 mL of 50% v/v resin slurry gives a ~1 mL bed, so 1 CV = 1 mL (e.g., 10 CV = 10 mL). Specifying buffer volumes by CVs lets you scale the purification up or down and use the same protocol.
Purify Protein¶
Load lysate: Load up to 10 CV (10 mL) clarified lysate per column. Optionally capture flowthrough for later analysis.
Note: if you have >10 mL lysate, load in tranches.
If you have more than 10 mL of clarified lysate, you can load your sample in tranches of 10 mL at a time. At a point, your resin bed will saturate and you will stop binding more protein. You can determine this saturation point by analyzing the flowthrough of each tranche by protein gel. If you are saturating your columns, consider using a larger resin bed.
Wash: Wash each column with ≥ 10 CV (10 mL) cold Wash Buffer. Allow buffer to flow through. Optionally capture flowthrough for later analysis.
Elute: Elute sample with 6 CV (6 mL) cold Elution Buffer. Capture flowthrough in a 15 mL centrifuge tube.
Storing Columns¶
Short-term (up to 48 h): Seal each column with a cap and bung and store at 4 °C.
Long-term / reuse: To store columns longer than 48 h — or to save used columns for later reuse — store them in 20% EtOH. Wash the column in Wash Buffer (as above), then with 10 CV (10 mL) Ultrapure water to remove salts. Fill the column with 20% EtOH, seal the bung and lid with parafilm, and store at 4 °C.
Note: why store in EtOH?
Packed and loaded columns can harbor bacterial growth, even at 4 °C. Storing in 20% EtOH prevents this for columns you will not use within 48 h, or that you want to reuse later.
Downloads¶
Acknowledgments¶
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
- Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., & Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology, 19(8), 751–755. 10.1038/90802
- Lavickova, B., & Maerkl, S. J. (2019). A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System. ACS Synthetic Biology, 8(2), 455–462. 10.1021/acssynbio.8b00427
- Grasemann, L., Lavickova, B., Elizondo-Cantú, M. C., & Maerkl, S. J. (2021). OnePot PURE Cell-Free System. Journal of Visualized Experiments, 172. 10.3791/62625