Determining proteome-wide expression levels using reverse protein arrays in fission yeast

Nat Protoc. 2012 Oct;7(10):1830-5. doi: 10.1038/nprot.2012.114. Epub 2012 Sep 13.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Profiling / methods
  • Gene Library
  • Open Reading Frames
  • Protein Array Analysis
  • Proteome
  • Proteomics / methods*
  • Schizosaccharomyces / genetics*
  • Schizosaccharomyces pombe Proteins / genetics*
  • Schizosaccharomyces pombe Proteins / metabolism


  • Proteome
  • Schizosaccharomyces pombe Proteins