Les_6_ppt_blackwhite

Les_6_ppt_blackwhite - PPI 1. 2. 3. Introduction Epitope...

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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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3.8. Protein arrays •DNA microarrays: software, hardware and detection systems FL or domain analytical array (~ ELISA) Detection: Fl, lum,affinity, photochemical, RA tags coupled to probes functional array lysate (from diff tissues) Specific Ab Reverse phase array library probes
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Protein attachment 1. Random orientation: 1. amines - aldehyde/epoxy- treated slide 2. Passive adsorption: glass coated with nitrocellulose, gel pads or poly-L-Lys 2. Uniform orientation: 1. Affinity tags: His 6 – Ni 2+ slide Biot – Strep slide Slides, wells, beads
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Proteome library • Gateway cloning technique • High quality required! Problems: expression, purification, storage, stability SOLUTION: in situ proteome libraries • Commercial collections Mainly restricted to yeast: protein production in homologous systems (e.g. making of a library of yeast proteins by using yeast expression system)
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In situ proteome libraries 1. NAPPA (nucleic acid programmable protein array): Biot. DNA spotted : In situ production of recombinant proteins by IVTT Anti-GST Ab / Ni 2+ - GST/His 6 -X Query plasmid IVTT to query protein Y
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2. PISA (Protein in situ array) Non captured DNA IVTT to protein library Capture protein library onto slide 2. nano-well format Non captured DNA IVTT to protein library 1 protein /well non immobilized (keep conformation, activity,….!) DNA stays in well: not washed away!
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4. DAPA (DNA array to protein array ) DNA templates spotted + immobilized onto slide (Ni 2+ treated) protein array slide IVTT of DNA array: (His 6 -tagged) proteins captured on protein array +: DNA array slide can be re-used!
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+/- Rapid and automated (HT) Sensitive Low sample consumption Assays can be performed in parallel Some software and hardware tools shared with DNA microarrays in situ proteome libraries: IVTT protein production: cost, better maintenance of protein function, conformation, stability, no long-term storage, … technical challenges classical proteome libraries: high quality protein production and purification needed in situ proteome libraries In vitro system
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4. Genetic approaches biochemical genetic Pool of preys Prey analysis Proteins, cDNA (protein arrays) Ab s , MS cDNA library: gene available sequencing Bait: X Prey: Y
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4.1. Libraries Bait expressed and functional in chosen cDNA library! Classical cDNA libraries
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Les_6_ppt_blackwhite - PPI 1. 2. 3. Introduction Epitope...

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