Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2....

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 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)

FACS MachineFluorescence cells labeled with negative charges,Non-fluorescence cells with positive charges,Clustered cells no charges due to their large sizes

Laser capture microdissectionA laser beam to excise a region of interest and select for further culture

In vitro Cell CulturesSome 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. Centrifuge2. Chromatography3. Polyacrylamide Gel Electrophoresis4. Mass Spectrometry

The preparative Ultracentrifuge

Cell Fraction by Centrifuge

Further separationVelocity sedimentation

(size and shape) and equilibrium sedimentation

(buoyant density)

The separation of molecules by column

chromatography

Three Typical Chromatography Methods

Three Typical Chromatography MethodsAffinity 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

Western Blot

Separation of protein molecules by isoelectric focusing

2D PAGE Coomassie blue stainging

2D Western BlotTobacco 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)