Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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New Protein Extraction/Solubilization Protocol for Gel-based Proteomics of Rat (Female) Whole Brain and Brain Regions

  • Hirano, Misato (Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST) WEST) ;
  • Rakwal, Randeep (Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST) WEST) ;
  • Shibato, Junko (Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST) WEST) ;
  • Agrawal, Ganesh Kumar (Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB)) ;
  • Jwa, Nam-Soo (Department of Molecular Biology, College of Natural Science, Sejong University) ;
  • Iwahashi, Hitoshi (Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST) WEST) ;
  • Masuo, Yoshinori (Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST) WEST)
  • Received : 2006.04.06
  • Accepted : 2006.06.12
  • Published : 2006.08.31
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Abstract

The rat is an accepted model for studying human psychiatric/neurological disorders. We provide a protocol for total soluble protein extraction using trichloroacetic acid/acetone (TCA/A) from rat (female) whole brain, 10 brain regions and the pituitary gland, and show that two-dimensional gel electrophoresis (2-DGE) using precast immobilized pH (4-7) gradient (IPG) strip gels (13 cm) in the first dimension yields clean silver nitrate stained protein profiles. Though TCA/A precipitation may not be "ideal", the important choice here is the selection of an appropriate lysis buffer (LB) for solubilizing precipitated proteins. Our results reveal enrichment of protein spots by use of individual brain regions rather than whole brain, as well as the presence of differentially expressed spots in their proteomes. Thus individual brain regions provide improved protein coverage and are better suited for differential protein detection. Moreover, using a phosphoprotein-specific dye, ingel detection of phosphoproteins was demonstrated. Representative high-resolution silver nitrate stained proteome profiles of rat whole brain total soluble protein are presented. Shortcomings apart (failure to separate membrane proteins), gel-based proteomics remains a viable option, and 2-DGE is the method of choice for generating high-resolution proteome maps of rat brain and brain regions.

Keywords

Acknowledgement

Supported by : HSS

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