For the past twenty years, 2-D gel electrophoresis has been the predominant technique for analyzing the protein constituents of whole cells and cell organelles. 2-D gel separates proteins based on the charge or pKa, i.e., the acidity and alkalinity, of the protein molecules on one axis, and molecular size on the other axis. Individual proteins in the gel are isolated as “spots,” and further characterization of spots of interest may be carried out by excising the spot from the gel, and applying appropriate sample preparation procedures for analytical measurements.
This technique is notoriously difficult for many reasons. First, only a small fraction, a few percent, of the proteins in a cell show up as spots in the gel. Second, identifying the spot of interest in the gel in a typically extremely complicated spot pattern is difficult. Third, the protein to be isolated is tangled in the gel and needs to be removed from the gel by cutting it out and then dissolving in solvents. Fourth, reproducibility is poor, making it difficult to compare spots in two different gels. Fifth, the technique is extremely labor intensive, from pouring gels to finding differences among the numerous spots for different gels, to cutting and processing the separated proteins. Last but not least, quantitation of the proteins in the gel is nearly impossible.
The device and techniques described herein will overcome many of these difficulties.