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May be it would have been better if Patrick O'Farrell's technique for resolution of proteins by two dimensional electrophoresis was never published in view of the huge waste of effort generated by the repeated application of this method. At best, 6000 to 7000 protein spots can be resolved by combination of isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis. However, we now know that over 100,000 distinct protein isoforms are generated by alternative alternative splicing of the approximately 21,500 protein-coding genes specified by the human genome. Coupled with post-translation modifications, like protein phosphorylation, the number of distinct protein species rises by another one or two orders of magnitude. The addition of a single phosphate group to a protein can significantly shift its position to the left on these gels so as to become recognized as a total different protein spot. With common techniques for protein detection such as silver-staining or fluorescent dye labelling, 2D gels reveal primarily abundantly expressed proteins such a structural proteins or metabolic pathway enzymes. The more interesting regulatory proteins such as protein kinases and receptors, which are expressed at 100-fold or more lower levels, are almost never visible as they are usually obscured by the much more abundant house-keeping proteins. Running and analyzing 2D gels can take typically 3 days, and it is extremely difficult to obtain 2D gel images from replicate samples that are completely superimposable unless one reverts to differential 2D gel electrophoresis (DIGE), which results in decreased sensitivity as half of much of each protein sample is subjected to the 2D gel electrophoresis. At the very best, O'Farrell gels reveal a small fraction of the true complexity of the proteomes of cells and tissues.

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