Prepare Columns

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	Link
Columns
Gravity columns	Empty Disposable PD-10 Columns	Cytiva	17043501	$258.30	4C to 30C	[link]

Resin
Ni-His Resin	NEBExpress Ni Resin	New England Biolabs	S1428S	$358.00	1C to 4C	[link]

Buffers
Potassium Hydroxide	Potassium hydroxide - ACS reagent, ≥85%, pellets	Sigma-Aldrich	221473-1KG	$99.90	4C to 30C	[link]
Hydrochloric acid (1M)	Hydrochloric acid 1 N, Reagent Grade, Packaging=Amber Bottle, Size=500 mL	VWR	E447-500L	$72.46	4C to 30C	[link]
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]

Protocol

Prepare Stock Solutions and P1 Wash Buffer as specified in

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

Pack a filter into the bottom of the column using the back end of a cell spreader.

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Notes: the columns have ridges!

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 milliQ water; allow water to flow through.

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Notes: “CV” = Column Volume

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

Papers

Original PURE paper

Cell-free translation reconstituted with purified components

Nature Biotechnology - Cell-free translation reconstituted with purified components

doi.org

Cell-free translation reconstituted with purified components

OnePot PURE

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

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription–translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

doi.org

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

OnePot PURE Cell-Free System | Text Page

Scientific Article | The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for...

dx.doi.org

OnePot PURE Cell-Free System | Text Page

Credits

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

Developers