• Getting Started
• Making PURE
• 1. Prepare Buffers and Columns
• 2. Prepare Bacterial Lysates for Protein Purification
• 3. Purify Proteins
• 4. Run PURE Reactions
• Measuring PURE
• 1. Measure protein concentrations
• 2. Plate reader

Getting Started

PURE (”Protein Synthesis Using Recombinant Elements”) is a defined cell-free system capable of transcribing mRNAs (using the T7 RNA polymerase) and translating proteins (using E. coli ribosomes and translation factors). PURE is built from individually purified components — proteins, RNAs, organic molecules, etc. The protocols here cover expressing and purifying protein components, preparing energy solutions and buffers, and assembling a complete PURE reaction.

Making PURE

1. Prepare Buffers and Columns

Make Protein Purification Buffers and Media

Make Amino Acid Mix

Make Energy Mix

Prepare Columns

2. Prepare Bacterial Lysates for Protein Purification

Grow and Induce Expression Strains

Prepare Bacterial Lysates by Sonication

3. Purify Proteins

Perform Ni-His Purification by Gravity Column

Perform Buffer Exchange and Concentration

4. Run PURE Reactions

Assemble Protein Mix

Assemble PURE Reactions

Measuring PURE

1. Measure protein concentrations

BCA Assay

Protein Gel

Pierce660 Assay

2. Plate reader

Measuring Fluorescence with Plate Reader