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. Cell-Cell Junction: adherens junction, cadherins
Manipulating DNA, RNA and Proteins Isolation of cells and cell culture DNA manipulation Protein measurement and analysis
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 Centrifuge to separate based on size. 2. adhesion strength 3. Antibody binding A. FACS (fluorescence activated cell sorter). B. Microdissection (Laser capture microdissection)
FACS Machine Fluorescence cells labelled with negative charges, Non-fluorescence cells with positive charges, Clustered cells no charges due to their large sizes
Laser capture microdissection A laser beam to excise a region of interest and select for further culture
In vitro Cell Cultures Some terminologies: in vitro, in vivo, primary culture Phase contrast images of fibroblasts (A) and myoblasts (B)
In vitro Cell Cultures Phase contrast images of oligodentrocytes (C) and tobacco cells (BY2 immortal cell line) (D)
Hybrid Cells
Preparation of Hybridomas that secrete desired monoclonal antibodies
Cell Fraction 1.Centrifuge 2.Chromatography 3.Polyacrylamide Gel Electrophoresis 4.Mass Spectrometry
The preparative Ultracentrifuge
Cell Fraction by Centrifuge
Further separation Velocity sedimentation (size and shape) and equilibrium sedimentation (buoyant density)
The separation of molecules by column chromatography
Three Typical Chromatography Methods
Affinity Chromatography usually gives better specificity
Protein purification with the combination of chromatography approaches
SDS and b-mercaptoethanol for Electrophoresis
SDS PAGE I
SDS PAGE II
SDS PAGE III
Western Blot
Separation of protein molecules by isoelectric focusing
2D PAGE Coomassie blue stainging
2D Western Blot Tobacco cells, (A) gel staining (B) phospho-thereonine
A peptide map or fingerprint of a protein
Mass Spectrometry Matrix-assisted laser desorption ionization-time-of-flight spectrometry (MALDI-TOF)
Genetic Engineering
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
Restriction nucleases
Rejoining DNA after restriction digestion
Gel Electrophoresis to separate DNAs with different sizes
Labeling DNA in vitro (I)
Labeling DNA in vitro (II)
DNA hybridization
Nucleic acid hybridization for the determination of specific DNA fragment
Northern and Southern Blots RNA and DNA Detection
Northern and Southern Blots
Microarray
DNA Cloning
DNA cloning and amplification
DNA sequencing (I)
DNA sequencing (II)
DNA sequencing (III)
DNA sequencing (IV)
DNA sequencing (V)
Polymerase Chain Reaction (PCR) (I)
Polymerase Chain Reaction (PCR) (II)
Polymerase Chain Reaction (PCR) (III)
PCR Applications Amplification of a gene of interest for cloning
PCR Applications Forensic Science
Protein Production by DNA Cloning
Analyzing Protein Functions With DNA Cloning Tools
Localization or Amplification of proteins by DNA Cloning of fusion proteins
Detection of protein interactions by DNA Cloning of fusion proteins (I)
Detection of protein interactions by DNA Cloning of fusion proteins (II)
Detection of protein interactions by DNA Cloning of fusion proteins (III) Yeast two-hybrid system
Identify Tumor Biomarkers by DNA Cloning Phage display
Gene Mutation
Digestion and Destroy
Summary 1.Isolating Cells 2.Cell Fraction 3.Genetic Engineering 4.Protein analysis with DNA cloning tools