last class 1.junctions: occluding junctions, anchoring junctions, communicating junctions 2....
TRANSCRIPT
![Page 1: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/1.jpg)
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
![Page 2: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/2.jpg)
• Manipulating DNA, RNA and Proteins
• Isolation of cells and cell culture• DNA manipulation
• Protein measurement and analysis
![Page 3: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/3.jpg)
• 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)
![Page 4: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/4.jpg)
FACS MachineFluorescence cells labeled with negative charges,Non-fluorescence cells with positive charges,Clustered cells no charges due to their large sizes
![Page 5: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/5.jpg)
Laser capture microdissectionA laser beam to excise a region of interest and select for further culture
![Page 6: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/6.jpg)
In vitro Cell CulturesSome terminologies: in vitro, in vivo, primary culture
Phase contrast images of fibroblasts (A) and myoblasts (B)
![Page 7: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/7.jpg)
In vitro Cell Cultures
Phase contrast images of oligodentrocytes (C) and tobacco cells (BY2 immortal cell line)
(D)
![Page 8: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/8.jpg)
![Page 9: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/9.jpg)
Hybrid Cells
![Page 10: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/10.jpg)
Preparation of Hybridomas that secrete desired monoclonal
antibodies
![Page 11: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/11.jpg)
![Page 12: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/12.jpg)
Cell Fraction
1. Centrifuge2. Chromatography3. Polyacrylamide Gel Electrophoresis4. Mass Spectrometry
![Page 13: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/13.jpg)
The preparative Ultracentrifuge
![Page 14: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/14.jpg)
Cell Fraction by Centrifuge
![Page 15: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/15.jpg)
Further separationVelocity sedimentation
(size and shape) and equilibrium sedimentation
(buoyant density)
![Page 16: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/16.jpg)
The separation of molecules by column
chromatography
![Page 17: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/17.jpg)
Three Typical Chromatography Methods
![Page 18: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/18.jpg)
Three Typical Chromatography MethodsAffinity Chromatography usually gives better specificity
![Page 19: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/19.jpg)
Protein purification with the combination of chromatography
approaches
![Page 20: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/20.jpg)
![Page 21: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/21.jpg)
SDS and b-mercaptoethanol for Electrophoresis
![Page 22: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/22.jpg)
SDS PAGE I
![Page 23: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/23.jpg)
SDS PAGE II
![Page 24: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/24.jpg)
SDS PAGE III
![Page 25: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/25.jpg)
Western Blot
![Page 26: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/26.jpg)
Western Blot
![Page 27: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/27.jpg)
Separation of protein molecules by isoelectric focusing
![Page 28: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/28.jpg)
2D PAGE Coomassie blue stainging
![Page 29: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/29.jpg)
2D Western BlotTobacco cells, (A) gel staining (B) phospho-thereonine
![Page 30: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/30.jpg)
A peptide map or fingerprint of a protein
![Page 31: Last Class 1.Junctions: Occluding Junctions, Anchoring Junctions, Communicating Junctions 2. Occluding Junctions: Tight Junction 3. Anchoring Junctions:](https://reader033.vdocuments.mx/reader033/viewer/2022051018/56649ea85503460f94babd1b/html5/thumbnails/31.jpg)
Mass Spectrometry
Matrix-assisted laser desorption ionization-time-of-flight
spectrometry (MALDI-TOF)