Two-dimensional gel electrophoresis/liquid chromatography for the micropreparative isolation of proteins.
Although comprehensive, column-based two-dimensional separation techniques offer enormous resolving power for a complex mixture, they often lack the ability to isolate the separated species for further analysis (e.g., proteins for Edman sequencing). This paper describes a micropreparative two-dimensional separation system for the isolation of proteins from complex mixtures, such as cell lysates. The system is composed of commercially available equipment: continuous-elution tube gel electrophoresis as the first dimension followed by gradient elution, reversed-phase perfusion liquid chromatography as the second dimension with a two-loop sampling valve as the interface between dimensions. The two-dimensional electrophoresis/liquid chromatography system (2D-EP/LC) shows high resolution of proteins since each dimension has orthogonal separation mechanisms (electrophoresis, size/charge; LC, hydrophobicity). Identification of proteins for further analysis is accomplished by superimposing a grid on the computer-generated 3D image.
Rose
D J
DJ
Department of Bionalytical and Structural Chemistry, Glaxo, Inc. Research Institute, Research Triangle Park, North Carolina 27709.
Opiteck
G J
GJ
D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D011506:Proteins / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured
D011506:Proteins