New protein extraction/solubilization protocol for gel-based proteomics of rat (female) whole brain and brain regions.
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 pre-cast 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, in-gel 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.
Hirano
Misato
M
Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology WEST, Ibaraki 305-8569, Japan.
Rakwal
Randeep
R
Shibato
Junko
J
Agrawal
Ganesh Kumar
GK
Jwa
Nam-Soo
NS
Iwahashi
Hitoshi
H
Masuo
Yoshinori
Y
D000818:Animals; D001923:Brain Chemistry; D005591:Chemical Fractionation / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D009419:Nerve Tissue Proteins