Les_5_complete_ppt_blackwhite

Les_5_complete_ppt_blackwhite - PPI 1. 2. 3. Introduction...

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PPI 1. Introduction 2. Epitope tagging 3. Biochemical approaches 1. in vitro interaction 2. Protein affinity chromatography 3. coIP 4. Crosslinking 5. TAP technology 6. SPA 7. Biacore 8. Protein arrays 4. Genetic approaches 1. cDNA libraries 2. Phage display 3. Y2H + variations 5. Summary 6. Validation small scale, single interactions large scale, interaction networks, screening General build-up:
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2. Epitope tagging Name Sequence #AA Origin Use α -tubulin B-tag E-tag c-Myc Flag HA His 6 His 10 HSV VSV-G T7 EEF QYPALT GAPVPYPDPLERP EQKLISEEDL DYKDDDDK YPYDVPDYA QPELAPEDPED YTDIEMNRLGK MASMTGGQQMG 3 6 13 10 8 9 6 10 11 11 11 C-terminal sequence of yeast protein Vp7 protein of blue-tongue virus Synthetic sequence Human c-myc gene protein Synthetic peptide Peptide form human influenza hemagglutinin protein Polyhistidine Polyhistidine Peptide form HSV glycoprotein D Vesicular stomatitis v glycoprotein Major capsid protein of phage T7 Binds metal ions ( affinity chromatography) (immuno)-affinity tags: -Ab recognition (exc: oligo-His, biotin, …) - fused C- or N-terminally to protein (linker) - multimers: Ab recognition - 6-30 AA long - synthetic or natural origin protein immunoreactive to a commercial Ab Sequential Conformational Epitopes: - portion of a molecule recognized by an Ab - linear or discontinuous -Sequential or conformational
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Procedure of tagging epitope CDS cloned in frame with protein ORF A. Epitope tagging via PCR B. Epitope tagging via adaptor duplex: 1. Hybridization of 2 synthetic complementary strands (CDS tag + RE) 2. Ligate adaptor into expression vector containing CDS of protein of interest Larger tags protein fused to * FP: fluorescence * CBP (calmodulin) , MBP (maltose) : Tandem affinity purification * GST (glutathion)
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Applications of tags • Subcellular localization, movement • Purification • PPI • Advantages: – Faster and cheaper than making own Abs – No effect on function of protein fusion proteins
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3. Biochemical approaches : 3.1. Intro 1. Protein affinity chromatography 2. coIP 3. Crosslinking 4. TAP technology 5. SPA 6. Biacore 7. Protein arrays small scale, single interactions large scale, interaction networks, screening mass spectrometry fluorescence False negatives: Cell lysis loss of PPI False positives: gain of PPI “sticky” proteins inherent to all PPI detection systems confirmation by different techniques!!
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Single or multiple interactions • X – Y : – interaction site/domain (mutagenesis) – Stimulus – time/place, relocalization,… • X(bait) – Y n (preys) : screening Identification/visualization: – Silver or Coomassie staining MS – RA labeled (e.g. 35 S, 32 P,…) – tags
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3.2. protein affinity chromatography 1. Purified protein (tagged-) X 2. Protein X immobilization (by use of tag) to resin of column 3. Load cell extract 4. Wash 5. Elute 6. Detect Y ( in vitro interaction!!!)
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Immobilization Pure (recombinant) protein protein X immobilized to matrix by affinity-tags
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Les_5_complete_ppt_blackwhite - PPI 1. 2. 3. Introduction...

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