last class 1.junctions: occluding junctions, anchoring junctions, communicating junctions 2....
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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 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)
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