Prototype for integrated two-dimensional gel electrophoresis for protein separation.
Two-dimensional gel electrophoresis practitioners have long waited for a fully automated system. This article presents an integrated platform that is capable of complete automation from sample introduction to spots detection. The strip gel for the first dimensional separation is fixed on the edge of a discrete planar stage before separation. A pair of platinum pin electrodes for isoelectric focusing (IEF) makes contact from underneath the stage. IEF is performed directly after rehydration and protein loading. After the first dimensional separation, sodium dodecyl sulfate (SDS) equilibration is done on the same stage without moving the gel. The IEF stage is then moved horizontally to couple with a precast second dimensional gel. The <0.5 mm gap between the two gels is filled with poly (ethylene oxide) solution. After SDS-polyacrylamide gel electrohporesis separation, a charge-coupled device camera is used to detect spots via protein native fluorescence excited by a Hg (Xe) lamp with the gel inside the running cell. Potential for full automation is demonstrated with 0.5 microg of Escherichia coli proteins on this miniaturized platform. More than 240 spots are detected in a total experiment time of <2.5 h.
Xu
Aoshuang
A
Ames Laboratory, US Department of Energy and Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Sluszny
Chanan
C
Yeung
Edward S
ES
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005453:Fluorescence; D011506:Proteins / Q000302:isolation & purification*
D011506:Proteins