Gel-eluted liquid fraction entrapment electrophoresis: an electrophoretic method for broad molecular weight range proteome separation.
Although well-established as a technique for protein purification, the application of continuous elution tube gel electrophoresis to proteome fractionation remains problematic. Difficulties associated with sample collection, particularly at the high mass range or at low sample loadings, continue to plague the technique. Furthermore, an upper mass limit is imposed as slow-moving higher molecular weight proteins are progressively diluted during the collection phase. In short, with current technology, effective separation over a broad mass range has not been achieved. In this work, we present improved techniques for continuous elution tube gel electrophoresis to accommodate broad mass range separation of proteins. Our device enables rapid partitioning of a proteome into discrete mass range fractions in the solution phase. High recovery is achieved at submicrogram to milligram sample loadings. We demonstrate comprehensive, reproducible separations of protein mixtures, as well as separation of a proteome in as fast as 1 h, over mass ranges from below 10 to 250 kDa. Finally, we identified proteins from a prefractionated standard protein mixture using liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis.
Tran
John C
JC
Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, Nova Scotia, Canada B3H 4J3.
Doucette
Alan A
AA
D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D004586:Electrophoresis / Q000379:methods*; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification*; D015203:Reproducibility of Results
D001426:Bacterial Proteins; D020543:Proteome