Exchange Buffers and Concentrate by Spin Filtration

Getting Started

You have a sample of a biomolecule (e.g., T7RNAP, tRNA, ribosomes, plasmid, etc.) that is either (1) in the wrong buffer (e.g., has 500 mM Imidazole) or (2) is too dilute.

You can use centrifugal spin filters to concentrate your sample and change its buffer. These filters allow buffer and small molecules to flow through them, retaining large molecules bigger than the filter’s specified molecular weight.

Materials and Equipment

We use two different volume spin filters, depending on how much sample volume we need to process: 15 mL or 0.5 mL.

Name	Product	Manufacturer	Part #	Price	Storage Conditions	Link
15 mL spin filters (3 kDa cutoff)	Amicon® Ultra Centrifugal Filter, 3 kDa MWCO	Millipore Sigma	UFC9003	$125	4C to 30C	[link]
0.5 mL spin filters (3 kDa cutoff)	Amicon® Ultra Centrifugal Filter, 3 kDa MWCO	Millipore Sigma	UFC5003	$63	4C to 30C	[link]

Protocol

Dilute your sample ~10x with your new buffer (e.g., add 54 mL P3 Exchange Buffer to 6 mL eluted protein).

Load your sample onto a centrifugal filter column.

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Notes: use filter cutoffs 3x smaller than your sample.

Centrifuge samples at either 4000 rcf (4 mL or 15 mL filters) or 14 000 rcf (0.5 mL filters) at 4C until you’ve reached your target volume. Check on your sample volume in the first 10 min, then again as needed.

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Notes: if you have too much sample, load in tranches.

Continue to dilute and spin your sample, noting your sample volume and dilution volume at each step, until you’ve reached your target dilution factor (we recommend ≥ 300x).

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Notes: how to calculate dilution factor.

Store columns for later use.

Wash columns by loading with ddH2O and spinning.

Load columns with EtOH 20% (v/v) and store at room temp.

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Notes: If you see precipitate, dilute your sample immediately.

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