Two-dimensional gel electrophoresis (2DGE) is a multi-step procedure that can be used to separate hundreds to thousands of proteins with extremely high resolution. It works by separating proteins by their pI's in one dimension,using an immobilized pH gradient (first dimension: isoelectric focusing), and then by their MW's in the second dimension using SDS PAGE. Gel density in the SDS second dimension can be of a fixed value between 8% and 12% gel or a gradient (7.5 - 15%). The resulting gel is then stained using either Coomassie Blue or silver stain so the protein spots can be visualized. In our lab, the gels are then scanned using a GS-800 calibrated densitometer from which one can obtain TIFF files of the image. Image analysis is then performed using software that permits the comparison and analysis of replicate gels to determine which spots are statistically and scientifically meaningful. Proteins of interest can then be excised from the gel, characterized and identified by mass spectrometry.
Our lab uses 2DGE to analyze proteins from a number of mouse cell lines. To do this, we use Invitrogen’s Zoom IPG Runner System. The Zoom IPG Runner System includes high voltage electrophoresis core and lid assembly that fits a mini-cell chamber identical to the XCell SureLock mini-cell. The mini-cell holds two Zoom IPGRunner Cassettes. Because the IEF component is divided into 5 separate chambers, the Zoom system allows complex protein mixture to be separated into 5 discrete fractions. We tested the fractionation using fetal bovine serum and found the albumin in one fraction only; a big improvement over solution phase IEF methods that have no physical barriers between the fraction chambers. While the literature that accompanies the Zoom system recommends fractionating 1-2 mg total protein, we have had success fractionating as little as 150ug of protein.
To use the system, a complex protein mixture is loaded in the middle of the left side of the IEF strip, where the pH is neutral, and a voltage is applied across it. The proteins then migrate until they reach their isoelectric point. The gel is then soaked in a denaturing solution containing SDS and the strips are placed in the well of an SDS PAGE gel. The gels that come with the system are designed to accommodate the IEF strips easily. The SDS PAGE gels are then run for 17 hours. Following this, the gel is then ready to be stained and analyzed.
I have used the Zoom IPG Runner System for two years and have found it to be extremely useful. The fact that it can effectively fractionate as little as 150 – 200ug of total protein has made this system invaluable in our lab.
Ali A. Latiff, Ph.D.
Post Doctoral Fellow
Case Western Reserve University