A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization
✍ Scribed by Megan Choi; Robert A. Nordmeyer; Earl Cornell; Ming Dong; Mark D. Biggin; Jian Jin
- Publisher
- John Wiley and Sons
- Year
- 2010
- Tongue
- English
- Weight
- 347 KB
- Volume
- 31
- Category
- Article
- ISSN
- 0173-0835
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✦ Synopsis
Abstract
To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A “Counter Free‐Flow” elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high‐resolution separation of a complex protein mixture can be achieved on this system using SDS‐PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48–96 fractions over a mass range of ∼10–150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 μL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 μg per channel and reduced resolution.