1. Last Class 1.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 Ca 2+. 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)

9. Hybrid Cells

10. Preparation of Hybridomas that secrete desired monoclonal antibodies

12. Cell Fraction 1.Centrifuge 2.Chromatography 3.Polyacrylamide Gel Electrophoresis 4.Mass Spectrometry

13. The preparative Ultracentrifuge

14. Cell Fraction by Centrifuge

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

16. The separation of molecules by column chromatography

17. Three Typical Chromatography Methods

18. Affinity Chromatography usually gives better specificity

19. Protein purification with the combination of chromatography approaches

21. SDS and b-mercaptoethanol for Electrophoresis

22. SDS PAGE I

23. SDS PAGE II

24. SDS PAGE III

25. Western Blot

27. Separation of protein molecules by isoelectric focusing

28. 2D PAGE Coomassie blue stainging

29. 2D Western Blot Tobacco cells, (A) gel staining (B) phospho-thereonine

30. A peptide map or fingerprint of a protein

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