Spot volume vs. amount of protein loaded onto a gel: A detailed, statistical comparison of two gel electrophoresis systems

Abstract

The long‐term goal of this research program is to clarify the molecular mechanisms that participate in the formation of human pituitary macroadenomas. One approach to that goal is to characterize the differentially expressed proteins that are found by a comparison of the proteomes of control pituitary vs. macroadenoma tissues. In order to accurately perform a comparative proteomics study, based on the combination of two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) and PDQuest 2‐D analysis software, a reproducible 2‐DE separation system with a wide linear dynamic measure range is needed. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180×245×0.5 mm); a typical vertical system is the Dodeca system that analyzes up to 12 gels at a time on a single‐concentration gel (190×205×1.0 mm). We have evaluated (Zhan and Desiderio, Electrophoresis 2003, 24, 1834–1846) the spatial and quantitative reproducibility of the two second‐dimensional gel systems to separate a human pituitary proteome; that study showed a higher reproducibility for the Dodeca gel system. This present study investigated the relationship between the spot volume and the amount of protein loaded onto the gel for those two 2‐D systems. The results demonstrated that the Dodeca gel system provides a wider linear dynamic range to measure the changes in the protein abundance in pituitary proteome.