Read the research paper global analysis of protein

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6-14. Read the research paper, “Global analysis of protein localization in budding yeast” Nature. 2003 Oct 16;425:686-91. A PDF version of the paper is available on the class website. 6. What is a proteome? The proteome is the entire network of protein and protein complexes inside the cell. 7. What advantages do genomic GFP fusions have over other methods of protein localization, such as over-expression of gene fusions or immunofluorescence? (choose all that apply in any order). A. GFP can be viewed using a light microscope B. GFP can be viewed using a fluorescence microscope C. GFP can be viewed using an electron microscope D. GFP can be viewed in live cells E. GFP can be viewed in fixed cells F. Genomic GFP fusions are not overexpressed G. Its difficult to make antibodies against the whole proteome ANSWER: BDFG. Genomic GFP fusions eliminate the need to over- express GFP fusions, resulting in possible mis-localization due to abnormally high levels of the fusion protein in the cell. Because GFP can be visualized in living cells, it is not necessary to expose cells to damaging treatments that might perturb localization patterns (i.e., fixation techniques). 8. Why did the authors combine their GFP fusion libraries with RFP fusions? RFP fusions fluoresce at a different wavelength and were fused to proteins with known localizations. This set of strains served to help co- localize GFP fusions that were difficult to assign. Thus, in the same strain, both the unknown (GFP) and a standard (RFP) could be compared.
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