Getting Started
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.
Materials and Equipment
Name | Product | Manufacturer | Part # | Price | Storage Conditions |
Columns | |||||
Columns | Empty Disposable PD-10 Columns | Cytiva | 17043501 | $258.30 | 4C to 30C |
Resin | |||||
Ni-His Resin | NEBExpress Ni Resin | New England Biolabs | S1428S | $358.00 | 1C to 4C |
Buffers | |||||
Potassium Hydroxide | Potassium hydroxide - ACS reagent, ≥85%, pellets | Sigma-Aldrich | 221473-1KG | $99.90 | 4C to 30C |
Hydrochloric acid (1M) | Hydrochloric acid 1 N, Reagent Grade, Packaging=Amber Bottle, Size=500 mL | VWR | E447-500L
| $72.46 | 4C to 30C |
HEPES | HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle | Sigma-Aldrich | H3375-500G | $431 | 4C to 30C |
Imidazole | ReagentPlus® Imidazole, Crystalline, 99% | Sigma-Aldrich | I202-500G | $120 | 4C to 30C |
Ammonium Chloride | Ammonium chloride,ACS reagent, ≥99.5% | Sigma-Aldrich | 213330-500G | $73.50 | 4C to 30C |
Sodium Chloride | Sodium Chloride, Redi-Dri™, anhydrous, free-flowing, ACS, ≥99% | Sigma-Aldrich | 746398-1KG | $133.00 | 4C to 30C |
Magnesium Chloride hexahydrate | BioXtra Magnesium chloride, Hexahydrate, Powder or Crystals, ≥99.0% | Sigma-Aldrich | M2670-100G | $50.50 | 4C to 30C |
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 |
Protocol
Prepare Stock Solutions and P1 Wash Buffer as specified in 
Make Protein Purification Buffers and Media
Make Protein Purification Buffers and Media Pack columns.
We used PD-10 gravity columns, which came with ~ 12 mL gravity columns, plastic filters (to hold the resin bed), plastic bungs (caps for column tips) and plastic lids (caps for column openings).
Cut column tips off using scissors or razor blade
For each column, first pack a filter into the bottom of the column using the back end of a cell spreader
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Wash empty columns.
Attach columns to a lab stand by clamp above a beaker (≥ 500 mL) to capture waste flowthrough.
Wash columns and filters with 5 CV (of water (milliQ); allow water to flow through.
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Load and Equilibrate Ni-His Resin into Ni-His columns.
Resuspend Ni-His resin (50% v/v suspension) by shaking bottle.
Add 2 mL resin by pipette to each column.
Equilibrate columns with 10 CV (20 mL) of cold P1 Wash Buffer (4C); allow buffer to flow through.
Store Columns
Seal each column with a cap and bung.
Store columns at 4C until ready for use.
Resources and References
Other Protocols
Papers
Resources
Credits
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
Developers
Testers