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

• Published in: Proteomics (Weinheim) 2002-06, Vol.2 (6), p.666-672
• Main Authors: Oguri, Takashi, Takahata, Iku, Katsuta, Kazuhiro, Nomura, Eiko, Hidaka, Masahiro, Inagaki, Naoyuki
• Format: Article
• Language: English
• Subjects: Animals; Cells, Cultured; Electrophoresis, Gel, Two-Dimensional - instrumentation; Electrophoresis, Gel, Two-Dimensional - methods; Hippocampus - cytology; Hydrogen-Ion Concentration; Low abundance; Narrow pH range immobilized pH grandients; Neurons - cytology; Proteome - chemistry; Rats; Resolution; Silver Staining - methods; Time Factors; Two-dimensional gel electrophoresis
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/1615-9861(200206)2:6<666::AID-PROT666>3.0.CO;2-V

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.