Description:

A rapid method for gene expression analysis, PURExpress^® is a novel cell-free transcription/translation system reconstituted from the purified components necessary for E. coli translation. With minimal nuclease and protease activity, the PURExpress system preserves the integrity of DNA and RNA templates/complexes and results in proteins that are free of modification and degradation. Transcription and translation are carried out in a one-step reaction, and require the mixing of only two tubes. With results available in a few hours, PURExpress saves valuable laboratory time and is ideal for high throughput technologies.PURExpress CitationsFigure 1: Protein expression using the PURExpress® In Vitro Protein Synthesis Kit.25 μl reactions containing 250 ng template DNA and 20 units RNase Inhibitor were incubated at 37°C for 2 hours. 2.5 μl of each reaction was analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703 ).Figure 2: Incorporation of 35S-methionine enables visualizationof protein by autoradiography.25 μl reactions containing 250 ng template DNA, 20 units RNase Inhibitor and 2 μl 35S-met were incubated at 37°C for 2 hours. 2.5 μl of each reaction was analyzed by SDS-PAGE, the gel was fixed for 10 minutes, dried for 2 hours at 80°C and exposed to x-ray film for 5 hours at -80°C.Figure 3: Schematic diagram of protein synthesis and purification by PURExpress.Figure 4: Expression and reverse purification of DHFR (A) and T4 DNA Ligase (B) using PURExpress.125 μl reactions were carried out according to recommendations in the accompanying manual. Samples were analyzed on a 10–20% Tris-glycine gel and stained with Coomassie Blue. Note that in both cases, the desired protein can be visualized in the total protein fraction. The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703 ).

Kit Components

The following reagents are supplied with this product:

	Store at (°C)	Concentration
RF1 (10 μl)
RF2 (10 μl)
RF3 (10 μl)
Control (DHFR) template
PURExpress Solution A
PURExpress Solution B		3.3X

Notes:

For a positive control reaction, use 2 μl of the supplied DHFR control template and 0.5 μl each of the supplied release factors.Each kit contains sufficient reagents for 10 x 25 µl reactions.The three release factors are supplied separately, allowing users to perform a protein synthesis reaction/ribosome display experiment with/without release factors of their choice.Release factors have not been added to solution B. You may still observe translational termination at a reduced level depending on your application and protein template design.PURExpress DHFR Control Template sequence files: Fasta, GenBank