title: Sample preparation and 2-DE procedure for protein expression profiling of black microcolonial fungi.
abstract: The ecology and stress adaptation of black rock inhabiting fungi in hot and cold extreme environments are not yet well understood. Two-dimensional gel electrophoresis (2-DE) is a promising tool to study the protein expression profiling and the metabolic status of microorganisms under stress conditions. The sample preparation has been shown to be the bottleneck for high resolution protein separation in 2-DE. For this purpose conditions must be optimized to obtain reliable and reproducible results. In addition, due to a multilayered and strongly melanized cell wall of black microcolonial fungi, special protocols for cell disruption and processing are required. In the present study, the protocol for protein extraction was established and optimized for the black yeast Exophiala jeanselmei MA 2853. The same protocol was successfully examined also for the meristematic fungus Coniosporium perforans MA 1299. Among the three procedures evaluated, trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation, and phenol extraction combined with methanol/ammonium acetate precipitation, the latter showed to be the best method for black yeasts and meristematic fungi. Penicillium chrysogenum was used as reference strain.
authors.affiliation: Department of Ecology and Sustainable Economic Development, University of Tuscia, Largo dell'Università snc, 01100 Viterbo, Italy. isola@unitus.it
authors.forename: Daniela
authors.initials: D
authors.lastname: Isola
authors.forename: Gorji
authors.initials: G
authors.lastname: Marzban
authors.forename: Laura
authors.initials: L
authors.lastname: Selbmann
authors.forename: Silvano
authors.initials: S
authors.lastname: Onofri
authors.forename: Margit
authors.initials: M
authors.lastname: Laimer
authors.forename: Katja
authors.initials: K
authors.lastname: Sterflinger
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000379:methods*; D001203:Ascomycota / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005093:Exophiala / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010408:Penicillium chrysogenum / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins