Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:

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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

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 labeled 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)