MicroPrep: Chip-based dielectrophoretic purification of mitochondria
| Autor: | Simon Werner, Kai Fuchsberger, Thanh Tu Duong, Meike Moschallski, Martin Stelzle, Britta Hagmeyer, Brigitte Angres, Dieter Stoll, Anton Posch, Monika Hausmann, Nancy Kunz, Aran Paulus, Heiko Steuer |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Chromatography
Chemistry Clinical Biochemistry Microfluidics Equipment Design Dielectrophoresis Cell Fractionation Biochemistry Mitochondria Analytical Chemistry Electrophoresis Microchip Mitochondrial Proteins Blot Cell Line Tumor Organelle Fluorescence microscope Humans Fluidics Centrifugation Cell fractionation |
| Zdroj: | ELECTROPHORESIS. 31:2655-2663 |
| ISSN: | 1522-2683 0173-0835 |
| Popis: | We have developed a microfluidic system - microPrep - for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long-term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging. Hence, microPrep yields tens of micrograms of enriched and purified mitochondria within hours. Western blots of mitochondria fractions showed that contaminating endoplasmatic reticulum was reduced by a factor 6 when compared with samples prepared by state of the art centrifugation. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text