Last Class

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• 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

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

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• 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)

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FACS Machine Fluorescence cells labeled with
negative charges, Non-fluorescence cells with
positive charges, Clustered cells no charges due
to their large sizes
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Laser capture microdissection A laser beam to
excise a region of interest and select for
further culture
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In vitro Cell Cultures Some terminologies in
vitro, in vivo, primary culture
Phase contrast images of fibroblasts (A) and
myoblasts (B)
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In vitro Cell Cultures
Phase contrast images of oligodentrocytes (C) and
tobacco cells (BY2 immortal cell line) (D)
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Hybrid Cells
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Preparation of Hybridomas that secrete desired
monoclonal antibodies
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Cell Fraction

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

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The preparative Ultracentrifuge
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Cell Fraction by Centrifuge
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Further separation Velocity sedimentation (size
and shape) and equilibrium sedimentation (buoyant
density)
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The separation of molecules by column
chromatography
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Three Typical Chromatography Methods
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Three Typical Chromatography Methods Affinity
Chromatography usually gives better specificity
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Protein purification with the combination of
chromatography approaches
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SDS and b-mercaptoethanol for Electrophoresis
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SDS PAGE I
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SDS PAGE II
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SDS PAGE III
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Western Blot
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Western Blot
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Separation of protein molecules by isoelectric
focusing
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2D PAGE Coomassie blue stainging
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2D Western Blot Tobacco cells, (A) gel staining
(B) phospho-thereonine
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A peptide map or fingerprint of a protein
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Mass Spectrometry Matrix-assisted laser
desorption ionization-time-of-flight spectrometry
(MALDI-TOF)
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Genetic Engineering
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Key Techniques

1. Restriction nucleases
2. DNA replication by vector or polymerase chain
   reaction
3. Accurate Nucleic acid hybridization
4. Gene sequencing
5. Monitoring Gene expression

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Gel Electrophoresis to separate DNAs with
different sizes
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Labeling DNA in vitro (I)
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Labeling DNA in vitro (II)
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DNA hybridization
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Nucleic acid hybridization for the determination
of specific DNA fragment
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Northern and Southern Blots RNA and DNA Detection
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Northern and Southern Blots
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Microarray
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DNA sequencing (I)
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DNA sequencing (II)
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DNA sequencing (III)
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DNA sequencing (IV)
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DNA sequencing (V)
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Polymerase Chain Reaction (PCR) (I)
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Polymerase Chain Reaction (PCR) (II)
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Polymerase Chain Reaction (PCR) (III) Movie
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PCR Applications Amplification of a gene of
interest for cloning
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PCR Applications Forensic Science
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Restriction nucleases
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Rejoining DNA after restriction digestion
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DNA Cloning
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DNA cloning and amplification
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Protein Production by DNA Cloning
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Analyzing Protein Functions With DNA Cloning Tools
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Localization or Amplification of proteins by DNA
Cloning of fusion proteins
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Detection of protein interactions by DNA Cloning
of fusion proteins (I)
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Detection of protein interactions by DNA Cloning
of fusion proteins (II)
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Detection of protein interactions by DNA Cloning
of fusion proteins (III) Yeast two-hybrid system
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Identify Tumor Biomarkers by DNA Cloning Phage
display
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Gene Mutation
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Gene Mutation
Digestion and Destroy
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Summary

1. Isolating Cells
2. Cell Fraction
3. Genetic Engineering
4. Protein analysis with DNA cloning tools