Proteome analysis of rat hippocampal neurons by multiple large gel two-dimensional electrophoresis

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Abstract
The goal of the present study was to detect as many protein spots as possible in mammalian cells using two-dimensional gel electrophoresis (2-DE). For proteome analysis, it is of importance to reveal as many proteins as possible. A single standard 2-DE gel (pH 3–10, 18 cm×20 cm, 13.5% gel) could detect 853 spots from proteins of cultured rat hippocampal neurons when visualized by silver staining. To increase the resolution of the separation and the number of detectable proteins by 2-DE, we utilized seven different narrow pH range immobilized pH gradients in the first dimension. In the second dimension, fourteen long SDS polyacrylamide gels were used: seven 7.5% gels for the separation of high molecular mass proteins (≥ 40 kDa) and seven 13.5% gels for the separation of low molecular mass proteins (≤ 40 kDa). Three hundred and sixty νg of proteins from cultured hippocampal neurons were loaded on to individual gels and visualized by silver staining. All 14 gel images were assembled into a 70 cm×67 cm cybergel that contained 6677 protein spots, thereby indicating that the utilization of the present strategy led to a 783% increase in the number of detected spots in comparison to the standard procedure. Loading double the amount (720 νg) of proteins on to a 13.5% gel led to a 184% increase in the number of detected spots, thereby indicating that the present strategy has a potential to display more protein spots in the cybergels.