The ePrep-System: A new electrophoretic approach for DNA isolation from biological samples.

We present a new free-flow electrophoretic separation system that extends the established concepts of nucleic acid migration in an electric field to a broadly applicable preparative scale. The system comprises a disposable flow tube in which the target nucleic acids are separated from impurities by a balanced combination of electrophoretic migration and counter-streaming electroosmotic flow under the influence of an applied external electric field. Despite the complex theoretical background the introduced electrophoretic technology offers simple hardware setup and handling protocols. A variable number of small and disposable flow tubes can be processed in parallel, which largely eliminates the cumulative increase in extraction times inherent to batch processing methods and allows faster throughput of intermediate sample numbers. We demonstrate easy isolation of nucleic acids without user interaction during the run by using existing and well established lysis chemistries. Sample loading is realized by concentrated transfer of DNA-loaded magnetic beads from a lysis reaction into the extraction flow tube. The present study centers on the development of a functional model for the device and the flow tube as well as a preliminary standard extraction protocol. The system is compatible with a broad range of sample types and we present proof of principle data demonstrating its suitability for biomarker detection in translational research applications.

Purification of GST-Tagged Proteins.

This protocol describes the purification of recombinant proteins fused to glutathione S-transferase (GST, GST-tagged proteins) by Glutathione Affinity purification. The GST tag frequently increases the solubility of the fused protein of interest and thus enables its purification and subsequent functional characterization. The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.g., agarose) and can be easily separated from a cell lysate by a bind-wash-elute procedure. GST-tagged proteins are often used to study protein-protein interactions, again making use of glutathione affinity in a procedure called a GST pull-down assay. The protocol is designed to process 200 ml of E. coli culture expressing intermediate to high amounts of a GST-tagged protein (~25 mg l(-1)). Depending on the expression rate or the available culture volume, the scale can be increased or decreased linearly. The protocol can also be used to purify GST-tagged proteins from other expression systems, such as insect or mammalian cells. Tips are provided to aid in modifying certain steps if proteins shall be recovered from alternative expression systems.