Two-Dimensional Gel Electrophoresis: Vertical Isoelectric Focusing

Abstract

Two-dimensional gel electrophoresis (2-DE) is one of the most powerful tools for separating proteins based on their size and charge. 2-DE is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical SDS-PAGE apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and, after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

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“…Protein samples were separated via standard SDS-PAGE or 2-DE that used vertical isoelectric focusing PAGE for the first dimension. 25 The gels were imaged with a LI-COR Odyssey imaging system (LI-COR, Inc, Lincoln, NE) with settings as a dual channel (700 and 800 nm; the 700-nm channel was monitored for visualizing the reference protein ladder), a focus offset of 0.75 mm for 1.5-mm-thick gel or 0.5 mm for 1.0-mm-thick gel, 7.0 intensity, and highest quality. In parallel, probe-reacted protein was also detected via Western blot.…”