Proteomic analysis of rat brain tissue: comparison of protocols for two-dimensional gel electrophoresis analysis based on different solubilizing agents.
The present study reports a comparison of recently described solubilizing methods, to set up a simple protocol for obtaining two-dimensional (2-D) gel electrophoresis maps of brain tissue. Different protocols were used for preparing rat brain homogenates and the resulting maps were compared by image analysis. Three different detergents, two delipidation methods, and introduction of a fractionation step based on different protein solubility in surfactants, were evaluated. When using efficient zwitterionic detergents (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, CHAPS; amidosulfobetaine 14, ASB-14), the patterns obtained by direct loading of total extracts were qualitatively overlapping with patterns obtained from fractionated samples. In contrast, a weaker nonionic agent (Nonidet P-40, NP-40) produced a different protein pattern in the collected fractions. Delipidation did not improve the results for all the different extraction methods. Immunoblots performed with antibodies recognizing cytosolic and membrane-spanning proteins, which were detected as nondegraded spots, showed that membrane proteins with intermediate molecular mass could be recovered. We suggest, as a simple and efficient method for preparing rat brain maps, the homogenization in a solution containing an efficient zwitterionic surfactant, which allows to solubilize cytosolic and membrane proteins in a single step. Alternatively, a fractionation can be carried out on samples homogenized by a weak solubilizing agent, a more labor-intensive effort resulting in a larger number of proteins on two maps.
Carboni
Lucia
L
Department of Molecular Medicine, Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline S.p.A., Verona, Italy. luc15324@gsk.com
Piubelli
Chiara
C
Righetti
Pier Giorgio
PG
Jansson
Birger
B
Domenici
Enrico
E
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001923:Brain Chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D008297:Male; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D020543:Proteome; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility
D009419:Nerve Tissue Proteins; D020543:Proteome