title: Determining proteome-wide expression levels using reverse protein arrays in fission yeast.
abstract: Global protein expression profiling of various mutants or growth conditions is currently a major challenge in biology. Here we provide a protocol for a strategy that we recently developed that couples ORFeome-based (ORF denotes open reading frame) expression to reverse protein arrays; this approach accurately quantifies more than 99% of the predicted fission yeast proteins in various genetic backgrounds. The first stage of this two-stage protocol requires mass mating between any fertile fission yeast mutant of interest and the integrated fission yeast-tagged ORFeome followed by selection of recombinant haploids. The second stage of the protocol, called reverse protein arrays, involves simple large-scale extraction of total proteins, which are then spotted on nitrocellulose membranes for detection by quantitative dot blot. When handled manually, the entire protocol takes about 2 months. However, the process could easily be automated and should also be applicable to other organisms.
authors.affiliation: Namur Research College, The University of Namur, Namur, Belgium.
authors.forename: Fanelie
authors.initials: F
authors.lastname: Bauer
authors.forename: Akihisa
authors.initials: A
authors.lastname: Matsuyama
authors.forename: Minoru
authors.initials: M
authors.lastname: Yoshida
authors.forename: Damien
authors.initials: D
authors.lastname: Hermand
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods; D015723:Gene Library; D016366:Open Reading Frames; D040081:Protein Array Analysis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D020543:Proteome; D029702:Schizosaccharomyces pombe Proteins