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CBNS 101 lecture2 - Suggested readings Lecture 1 Molecular...

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Suggested readings: Lecture 1: Molecular Biology of the cell, Alberts et al Visualizing cell (Microscopy): Chapter 9; Page 579-615 Universal features of cells and cell diversity: Chapter 1; page 3-39 Lecture 2 : Molecular Biology of the cell, Alberts et al Visualizing Cells (Microscopy): Chapter 9; Page 579-615 Membrane structure and function: Chapter 10; page 617-629 Lecture 3 : channels, carriers, synaptic transmission Molecular Biology of the cell, Alberts et al: Chapter 11:page 629-650
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Fluorescent dyes and fluorescent proteins
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Jablonski diagram Stokes shift
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Two different ways of labeling proteins 1. Fluorescent dyes: a) Fluorescein isothiocyanate (FITC) Excitation: 490nm; Emission: 520nm b) Rhodamine isothiocyanate (RITC) Excitation: 570nm; Emission: 595nm c) Cy5 Excitation: 650nm; Emission:667nm 2. Fluorescent proteins: a) Green fluorescent protein (GFP) Excitation: 489nm; Emission:508nm b) Blue fluorescent protein (BFP) Excitation: 380nm; Emission: 440nm c) Cyan fluorescent protein (CFP) Excitation: 434nm; Emission:477nm d) Red Fluorescent proteon (RFP) Excitation: 558nm; Emission:583nm
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Visualizing proteins 1. Immunohistochemistry 2. GFP-tagging of proteins Immunohistochemistry: Dyes can be attached to antibodies Target protein to be visualized Antibody against target protein Antibody is conjugated with a fluorescent dye
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GFP tagging of proteins Live cell imaging is possible: Protein dynamics within a cell can be Followed in real time
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Transgenic pigs expressing GFP protein
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Dark field microscopy 1. Principle of fluorescence emission 2. Fluorescent dyes 3. Fluorescent proteins 4. Tagging of proteins with dyes or fluorescent proteins 5. Fluorescence microscope 6. Confocal microscope 7. Three dimensional reconstruction of cells
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Membrane structure and function
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Membranes are dynamic structures with specific functions Import/export of specific molecules Regulate interaction between cells Regulate interactions between cell/environment Transfer information from outside to cell interior Define mechanical properties-movement, shape, growth
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Eucaryotic cells contain internal membrane-bound organelles Compartmentalization of distinct intra-cellular activities
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Cell membranes are crucial for life of a cell 1. Plasma menbrane encloses the cell, defines its boundaries maintains differences between cytosol and extarcellular space. 2. Within the cell, the membranes of ER, Golgi, mitochondria, choloroplast maintain their essential contents from that of cytosol. 3. Membranes contain specialized proteins which can synthesize ATP, the energy required to transport solutes, ions across cells. 4. Plasma membrane contains proteins which act as sensors to detect specific extracellular signals- receptors.
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THE LIPID BILAYER --Universal basis for membrane structure --Main constituents are lipids and proteins: Held together with non-covalent interactions --Lipids form an impermeable barrier to water soluble molecules, proteins act as carriers, sensors and binding agents with the extarcellular matrix --Continuous double layer-dynamic and fluid Electron micrograph
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