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Last Class Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions

Last Class Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions: adherens Junction 4. Communicating Junctions: Gap Junction 5. adherens junction, cadherins. Manipulating DNA, RNA and Proteins

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Last Class Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions

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  1. Last Class • Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions • 2. Occluding Junctions: Tight Junction • 3. Anchoring Junctions: adherens Junction • 4. Communicating Junctions: Gap Junction • 5. adherens junction, cadherins

  2. Manipulating DNA, RNA and Proteins • Isolation of cells and cell culture • DNA manipulation • Protein measurement and analysis

  3. Isolating Cell and Cell Culture • Tissue are disassembled by (1) preteolytic enzymes to separate cells from ECM, e.g. trypsin and collagenase, (2) together with reagents to chelate Ca2+. • 1. Centrifuge to separate based on size. • 2. adhesion strength • 3. Antibody binding • A. FACS (fluorescence activated cell sorter). • B. Microdissection (Laser capture microdissection)

  4. FACS Machine Fluorescence cells labeled with negative charges, Non-fluorescence cells with positive charges, Clustered cells no charges due to their large sizes

  5. Laser capture microdissection A laser beam to excise a region of interest and select for further culture

  6. In vitro Cell Cultures Some terminologies: in vitro, in vivo, primary culture Phase contrast images of fibroblasts (A) and myoblasts (B)

  7. In vitro Cell Cultures Phase contrast images of oligodentrocytes (C) and tobacco cells (BY2 immortal cell line) (D)

  8. Hybrid Cells

  9. Preparation of Hybridomas that secrete desired monoclonal antibodies

  10. Cell Fraction Centrifuge Chromatography Polyacrylamide Gel Electrophoresis Mass Spectrometry

  11. The preparative Ultracentrifuge

  12. Cell Fraction by Centrifuge

  13. Further separation Velocity sedimentation (size and shape) and equilibrium sedimentation (buoyant density)

  14. The separation of molecules by column chromatography

  15. Three Typical Chromatography Methods

  16. Three Typical Chromatography Methods Affinity Chromatography usually gives better specificity

  17. Protein purification with the combination of chromatography approaches

  18. SDS and b-mercaptoethanol for Electrophoresis

  19. SDS PAGE I

  20. SDS PAGE II

  21. SDS PAGE III

  22. Western Blot

  23. Western Blot

  24. Separation of protein molecules by isoelectric focusing

  25. 2D PAGE Coomassie blue stainging

  26. 2D Western Blot Tobacco cells, (A) gel staining (B) phospho-thereonine

  27. A peptide map or fingerprint of a protein

  28. Mass Spectrometry Matrix-assisted laser desorption ionization-time-of-flight spectrometry (MALDI-TOF)

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