A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization.
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 approximately 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 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution.
Choi
Megan
M
Genomics Division, Lawrence Berkeley National Laboratory, CA 94720, USA.
Nordmeyer
Robert A
RA
Cornell
Earl
E
Dong
Ming
M
Biggin
Mark D
MD
Jin
Jian
J
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D016969:Desulfovibrio vulgaris / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation* / Q000379:methods; D057166:High-Throughput Screening Assays; D013058:Mass Spectrometry; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013997:Time Factors
D001426:Bacterial Proteins; D011506:Proteins