Biology 2382B Lecture Notes - Lecture 3: Electric Field, Cell Membrane, Hydrophile
22 Mar 2018
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Isolation and analysis of cell organelles and molecules. Learning objectives: fluorescent activated cell sorting (facs, centrifugation for isolation of intracellular organelles, electrophoresis and western blotting for analysis of proteins. Cell cycle analysis by facs: cells that have replicated their dna but not fully divided (g2) will have twice the hoechst stain fluorescence intensity of non-dividing cells (g1) How to isolate cell organelles: disruption of cell plasma membrane, mechanical homogenization, sonication (ultrasound, pressure (force cells through narrow valve, non-ionic detergents i. e. triton x-100, placing cells in hypotonic solution, centrifugation of cell homogenate, differential, equilibrium density-gradient. ***safe centrifugation requires balanced loading of the centrifuge rotor imbalanced rotors can lead to damaged centrifuges and rotors. How are proteins separated from the organelle: detergents have hydrophobic and hydrophilic properties, non-ionic: reserve structure of protein but disrupt bilayer. Ionic: denature proteins: the negative chare in ionic detergents is responsible for bonding to & unfolding protein.
