Purify Proteins by Ni-His Gravity Column

Getting Started

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.

Materials and Equipment

Name	Product	Manufacturer	Part #	Price	Storage Conditions	Link
Columns
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]

Consumables
15 mL centrifuge tubes	Corning® 15 mL PP Centrifuge Tubes, Rack Packed with CentriStar™ Cap, Sterile, 50/Rack, 500/Case	Corning	430790	$230.44	4C to 30C	[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]
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]

Prerequisite Protocols

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.

Protocol

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 column volumes (”CVs”) of DI water (i.e., 5x 2mL resin bed = 10 mL DI water).

Equilibrate each column with ≥ 5 CVs (10 mL) cold P1 buffer.

Load up to 5 CVs (10 mL) clarified lysate per column and allow to flow through. (Optionally) capture flowthrough for later analysis; see

Pierce660 Assay and

Protein Gel.

‣

Notes: you can load more than 10 mL lysate in tranches.

Wash column with ≥ 5 CVs (10 mL) cold P1 buffer. Allow buffer to flow through. (Optionally) capture flowthrough for later analysis; see

Pierce660 Assay and

Protein Gel.

Elute sample in 2.5 mL cold P2 buffer. Capture flow through in 15 mL centrifuge tube.

‣

Notes: the first 500 uL fraction has little protein

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