Store Proteins

Getting Started

You have proteins and want to store them at -80C until you’re ready to use them. Here is how you do that.

Materials and Equipment

Name	Product	Manufacturer	Part #	Price	Storage Conditions	Link
Potassium Hydroxide	Potassium hydroxide - ACS reagent, ≥85%, pellets	Sigma-Aldrich	221473-1KG	$99.90	4C to 30C	[link]
HEPES	HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle	Sigma-Aldrich	H3375-500G	$431	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]
Glycerol	Glycerol, Molecular Biology Grade, Liquid, ≥99.0%	Sigma-Aldrich	G5516-1L	$157.00	4C to 30C	[link]
10 mL Luer lock syringes	Syringes with BD Luer-Lok® Tip, BD Medical, Syringe with Luer-Lok® Tip, Volume=10 mL	BD Industrial/Difco	550-80620-PK	$102.40	4C to 30C	[link]
0.22 um syringe filters	PES Syringe Filter, 0.22μm, 30mm, Sterile	CELLTREAT Scientific Products	229747	$84	4C to 30C	[link]

Protocol

Prepare Stock Solutions

Reagent	MW [g/mol]	Amount [g]	Final Volume [mL]	Storage	Needs pH adjustment?	Needs Sterilization?
Stock Solutions
Potassium Hydroxide (1M)	56.11	14.0	250	4C to 30C	no	no
HEPES-KOH (pH=7.6; 1M)	238.3	59.5	250	4C to 30C; dark	yes	no
Magnesium Chloride (1M)	203.3	20.3	100	4C to 30C	no	no

Make 4x P3 Concentrate - used to prepare P3 Exchange and P3 Storage Supplement.

Make P3 Storage Supplement - used to protect samples in P3 Exchange buffer from damage by freezing during storage at -80C. Add an equal volume of P3 Storage Supplement to samples in P3 Exchange Buffer (1:1). Filter P3 Storage Supplement with a 0.22 um syringe filter.

Reagent	Final Concentration [mM]
HEPES-KOH	50
Potassium Chloride	100
Magnesium Chloride	10
TCEP	1
Glycerol	60% (v/v)

Reagent	Volume to Add [mL]
4x P3 Concentrate	2.5
Glycerol	6.0
TCEP (0.5 M)	0.020
water (milliQ)	1.48
Total	10

Add an equal volume of P3 Storage Supplement to each protein sample (e.g., 100 uL P3 Storage Supplement to 100 uL EF-Tu in P3 Exchange Buffer).

Mix gently by pipetting.

Store frozen at -80C 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

Murray Lab