lecture25_slides

lecture25_slides - Overview of today Today: Go over Midterm...

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Overview of today Today: Go over Midterm #2 results Review protein-protein interaction networks More protein interaction network analysis
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Midterm #2 summary Median: 61/75 (81%) Std. dev: 12 Max: 71/75 >69 A >67 A- >65 B+ >62 B >60 B- >57 C+ >52 C >50 C- >47 D+ >40 D >35 D- (Out of 75)
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Protein-protein interactions support signaling
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Protein-protein interactions support structure Collins et al. Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature 2007, 446(7137):806-810.
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Introduction to protein-protein interaction networks Physical interactions between proteins are an important mechanism behind many cellular processes Methods of detection: Complex purification and protein identification (Mass spec. based) Binary detection experiments (Yeast 2-hybrid) Protein arrays
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Affinity purification A Protein of interest tag modification (e.g. HA/GST/His) this molecule will bind the ‘tag’.
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Affinity purification A the cell
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B Affinity purification A the cell naturally binding protein lots of other untagged proteins
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B Affinity purification A Ruptured membranes cell extract
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B Affinity purification A untagged proteins go through fastest (flow-through)
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B Affinity purification A tagged complexes are slower and come out later (eluate)
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Tandem affinity purification– mass spectrometry (TAP-MS) B A Complex pull- down/purification Mass spec. identification
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Yeast Two Hybrid Assay The two-hybrid system is a molecular genetic tool which facilitates the study of protein-protein interactions. If two proteins interact, then a reporter gene is transcriptionally activated. e.g. gal1-lacZ - the beta- galactosidase gene A color reaction can be seen on specific media. You can use this to Study the interaction between two proteins which you expect to interact Find proteins (prey) which interact with a protein you have already (bait).
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Two-hybrid assay 1. 2. 3. 4. A B Fields S. Song O. Nature. 1989 Jul 20;340(6230):245-6. PMID: 2547163 UASG GAL4-DBD SNF1 SNF4 Transcription activation domain Allows growth on galactose GAL1
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Yeast two hybrid a) DNA-binding and activation domains (circles) are fused to proteins X and Y. The interaction of X and Y leads to reporter gene expression (arrow).
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Hawoong Jeong et al. Oltvai Centrality and lethality of protein networks. Nature 411, 41-42 (2001) Yeast 2-hybrid interaction network
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Physical interaction detection caveats A lot of false positives/false negatives!! (low overlap between different datasets on the same organism under the same condition) Is an interaction A-B direct, or is it mediated by other proteins?
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lecture25_slides - Overview of today Today: Go over Midterm...

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