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Affinity chromatography/mass spec PowerPoint Presentation
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Affinity chromatography/mass spec

Affinity chromatography/mass spec

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Affinity chromatography/mass spec

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  1. Affinity chromatography/mass spec Bait protein GST Page 252

  2. Affinity chromatography/mass spec Bait protein GST Add yeast extract Protein complexes bind Most proteins do not bind Page 252

  3. Evaluation of affinity chromatography/mass spec Advantages: Thousands of protein complexes identified Functions can be assigned to proteins Disadvantages: False negative results False positive results Page 253-254

  4. Affinity chromatography/mass spec • False negatives: • Bait must be properly localized and • in its native condition • Affinity tag may interfere with function • Transient protein interactions may be missed • Highly specific physiological conditions • may be required • Bias against hydrophobic, and small proteins Bait protein GST Page 253

  5. Affinity chromatography/mass spec • False positives: • sticky proteins Bait protein GST Page 253

  6. The yeast two-hybrid system Bait protein DNA Binding Reporter gene Box 8-3 Page 255

  7. The yeast two-hybrid system Prey protein DNA activation Prey protein DNA activation Reporter gene Prey protein DNA activation Prey protein DNA activation Box 8-3 Page 255

  8. The yeast two-hybrid system Bait protein DNA Binding Prey protein DNA activation Reporter gene Box 8-3 Page 255

  9. The yeast two-hybrid system Bait protein DNA Binding Prey protein DNA activation Reporter gene Isolate and sequence the cDNA of the binding partner you have found Box 8-3 Page 255

  10. Evaluation of the yeast two-hybrid system Advantages: Thousands of protein complexes identified Functions can be assigned to proteins Disadvantages: Detects only pairwise protein interactions False-negative results (as for affinity chromatography) -- bait may be mislocalized -- transient interactions may be missed -- some complexes require special conditions -- bias against hydrophobic proteins False-positive results -- some proteins may be sticky -- bait protein may auto-activate a reporter Page 256

  11. The Rosetta Stone approach Marcotte et al. (1999) and other groups hypothesized that some pairs of interacting proteins are encoded by two genes in many genomes, but occasionally they are fused into a single gene. By scanning many genomes for examples of “fused genes,” several thousand protein-protein predictions have been made. Page 258

  12. The Rosetta Stone approach Yeast topoisomerase II E. coli gyrase B E. coli gyrase A Fig. 8.23 Page 256

  13. Pathway maps A pathway is a linked set of biochemical reactions ExPASy ProNet EcoCyc: E. coli pathways MetaCyc: 600 pathways, 300 organisms KEGG: Kyoto Encyclopedia of Genes & Genomes Issues: Is the extrapolation between species valid? Have orthologs been identified accurately? False positive, false negative findings Page 258

  14. Fig. 8.24 Page 257

  15. Fig. 8.24 Page 257

  16. Fig. 8.27 Page 260

  17. Fig. 8.29 Page 262

  18. Fig. 8.29 Page 262