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A multichannel gel electrophoresis and continuous fraction collection apparatus for high‐throughput protein separation and characterization
Author(s) -
Choi Megan,
Nordmeyer Robert A.,
Cornell Earl,
Dong Ming,
Biggin Mark D.,
Jin Jian
Publication year - 2010
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200900435
Subject(s) - chromatography , free flow electrophoresis , chemistry , fraction (chemistry) , elution , resolution (logic) , electrophoresis , two dimensional gel electrophoresis , gel electrophoresis , analytical chemistry (journal) , protein purification , gel electrophoresis of proteins , polyacrylamide gel electrophoresis , proteomics , biochemistry , computer science , artificial intelligence , gene , enzyme
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.