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

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. Cell-Cell Junction: 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 labelled 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 Centrifuge Chromatography Polyacrylamide Gel Electrophoresis 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. Three Typical Chromatography Methods 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

26. 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)

32. Genetic Engineering

33. Key Techniques Restriction nucleases DNA replication by vector or polymerase chain reaction Accurate Nucleic acid hybridization Gene sequencing Monitoring Gene expression

34. Restriction nucleases

35. Rejoining DNA after restriction digestion

36. Gel Electrophoresis to separate DNAs with different sizes

37. Labeling DNA in vitro (I)

38. Labeling DNA in vitro (II)

39. DNA hybridization

40. Nucleic acid hybridization for the determination of specific DNA fragment

41. Northern and Southern Blots RNA and DNA Detection

42. Northern and Southern Blots

43. Microarray

44. DNA Cloning

45. DNA cloning and amplification

46. DNA sequencing (I)

47. DNA sequencing (II)

48. DNA sequencing (III)

49. DNA sequencing (IV)

50. DNA sequencing (V)