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.
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Acknowledgements
We thank the GEMO laboratory for discussions. This work was supported by a European Molecular Biology Organization (EMBO) short-term fellowship (58-2008), and grants FRFC 2.4510.10, Credit aux chercheurs 1.5.013.09 and MIS F.4523.11 to D.H.; F.B. is a Fonds de la Recherche en Industrie et Agriculture (FRIA) Research Fellow; D.H. is a Fonds National de la Recherche Scientifique (FNRS; Belgium) Research Associate.
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F.B. designed and performed the experiments, and wrote the manuscript; A.M. and M.Y. designed the reverse protein array strategy, and helped with its development; D.H. designed the study and wrote the manuscript.
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Supplementary information
Supplementary Figure 1
Comparison of α-tubulin levels between the wild type and the mutant strains. Total extracts of biological triplicates of wild type and mutant (Δelp3Δctu1) strains have been collected and separated by PAGE; transferred onto nitrocellulose membrane, incubated with anti-α–tubulin primary antibodies, secondary fluorescent antibodies and scanned to measure the fluorescent signal of each lane. The resulting raw and mean values are indicated. (PDF 319 kb)
Supplementary Figure 2
A representative membrane after hybridization and scanning. Total extracts of wild type ORFeome were spotted in quadruplicates onto nitrocellulose membrane, incubated with anti-α–tubulin and anti-His6 primary antibodies, secondary fluorescent antibodies and scanned to measure the fluorescent signal of each dot. As expected, the level of α-tubulin does not show strong variation from one strain to another while tagged ORFeome proteins display a much broader range of expression level. (PDF 1452 kb)
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Bauer, F., Matsuyama, A., Yoshida, M. et al. Determining proteome-wide expression levels using reverse protein arrays in fission yeast. Nat Protoc 7, 1830–1835 (2012). https://doi.org/10.1038/nprot.2012.114
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DOI: https://doi.org/10.1038/nprot.2012.114
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