Home Resources Video Library Introduction to the NEBExpress® Cell-free Protein Synthesis System

Introduction to the NEBExpress® Cell-free Protein Synthesis System

Watch this tutorial explaining the streamlined workflow for our new NEBExpress® Cell-free Protein Synthesis System to learn how you can easily synthesize your protein in as little as 2 to 4 hours.

Script

The NEBExpress Cell-free E.coli Protein Synthesis System can synthesize protein in just 2 to 4 hours. This system includes all of the components needed for protein synthesis: a highly active cell extract, an optimized T7 RNA polymerase, an RNase inhibitor and a protein synthesis reaction buffer containing energy source, nucleotides, and amino acids. All that is required is a template containing the gene of interest under the control of a T7 RNA Polymerase promoter. The template DNA must contain: an in-frame start codon, an in-frame stop codon, a T7 promoter, an upstream ribosome binding site, a downstream spacer region, and a downstream T7 terminator. Template can be plasmid DNA, linear DNA, or mRNA. While higher yields are often obtained with a plasmid DNA, linear DNA or PCR products can generate acceptable yields and provide timesaving advantages. A PCR fragment can be synthesized using primers to add upstream and downstream elements such as the T7 promoter and terminator. All DNA or mRNA templates should have a complete T7 terminator or the minimal T7 stem/loop, as it will enhance mRNA stability. Template purity is also important. We recommend using a miniprep kit or a DNA cleanup kit to isolate plasmid or linear DNA. Once the template DNA is purified, thaw the NEBExpress Cell-free E. coli Protein Synthesis System components on ice. Gently vortex the NEBExpress S30 Synthesis Extract and Protein Synthesis Buffer to mix. Combine the following in a 1.5 ml microcentrifuge tube: 12 µl of NEBExpress S30 Synthesis Extract, 25 µl of 2X Protein Synthesis Buffer, 1µl of T7 RNA Polymerase, 1 µl of RNase Inhibitor, 250 ng of DNA template, and nuclease free water to total reaction volume of 50 µl. Incubate reactions at 37°C, with shaking, for 2 to 4 hours. The system routinely produces approximately 25 μg of protein per 50 μl reaction, or 1/2 mg/ml. Following incubation, the reaction can be stored frozen, or analyzed by your method of choice. For SDS-PAGE or Western blots, combine 2 µl of reaction with sample buffer and load directly on the gel. It is unnecessary to precipitate the sample with acetone or TCA. The components of this system do not interfere with gel electrophoresis. The synthesized protein can be isolated by affinity purification, for example, using NEBExpress Ni-NTA magnetic beads or NEBExpress Ni Spin columns for further analysis. To learn more, visit www.neb.com/E5360.

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