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Determining proteome-wide expression levels using reverse protein arrays in fission yeast
Fanelie Bauer1, Akihisa Matsuyama, Minoru Yoshida
1Namur Research College, The University of Namur, Namur, Belgium.
Nature Protocols
|September 15, 2012
Summary
This study presents a novel two-stage protocol for global protein expression profiling in fission yeast, accurately quantifying over 99% of predicted proteins using ORFeome-based expression and reverse protein arrays.
Area of Science:
- Proteomics
- Molecular Biology
- Yeast Genetics
Background:
- Global protein expression profiling is a significant challenge in biological research.
- Existing methods struggle to comprehensively analyze protein levels across diverse genetic backgrounds and conditions.
Purpose of the Study:
- To develop and present a robust protocol for accurate and comprehensive global protein expression profiling.
- To enable the quantification of a high percentage of predicted proteins in fission yeast.
Main Methods:
- A two-stage protocol combining ORFeome-based (open reading frame) expression with reverse protein arrays.
- Stage 1: Mass mating of fission yeast mutants with a tagged ORFeome library, followed by recombinant haploid selection.
- Stage 2: Large-scale total protein extraction and spotting onto nitrocellulose membranes for quantitative dot blot detection.
Main Results:
- Accurate quantification of over 99% of predicted fission yeast proteins.
- The protocol is effective across various genetic backgrounds.
- Manual execution takes approximately 2 months, with potential for automation.
Conclusions:
- The developed protocol offers a powerful strategy for comprehensive protein expression analysis.
- This method is adaptable for studying protein expression in different mutants and growth conditions.
- The approach shows promise for application in other organisms beyond fission yeast.
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