Biology 2382B Lecture Notes - Lecture 3: Hoechst Stain, Scatter Plot, Fluorophore

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3 isolation and analysis of cell organelles and molecules. Fluorescent activated cell sorting (facs: centrifugation for isolation of intracellular organelles, electrophoresis and western blotting for analysis of proteins. Individual cells are forced through a nozzle and given a charge proportional to the degree of fluorescence detected. Cell cycle analysis by facs: cells that have replicated their dna but not fully divided (g2) will have twice the. How to isolate cell organelles: procedures to break cells apart and not disrupt organelles inside while disrupting plasma membrane. Force cell through small valve so they burst open: non-ionic detergents i. e. , triton x-100, makes cells leaky, stuff comes out, placing cells in hypotonic solution, more water flows into cell due to osmosis and cells swell and ruptures. Step two: centrifugation of cell homogenate: differential, equilibrium density-gradient. Spinning requires a vacuum and cooling as it gets very hot: >100 000 g force --> wobbles, could break off rotator shaft and become projectile.

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