A Multichannel Gel Electrophoresis and Continuous Fraction Collection Apparatus for High-Throughput Protein Separation and Characterization

Abstract

To facilitate a direct interface between protein separation by polyacrylamide gel electrophoresis (PAGE) and protein identification by mass spectrometry, we developed a multi-channel 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-hour electrophoresis run, for example, each sample was separated and eluted into 48 to 96 fractions over a mass range of ~10 to 150 kD; 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 2-3 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.