Jan 11

Jan 11 - Microscopes & Proteomic Techniques...

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Microscopes & Proteomic Techniques Translational Science: from bench to bedside
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Fractionation Microarrays Two Hybrid Analysis Electron Microscopy
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Fig. 9-1
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Fractionation -cell plasma membrane must first be homogenized ” by: i)sonication ii) sheared iii) pressure iv) detergents i.e., Triton X-100 Then the contents separated by: Centrifugation i) Differential or ii) Equilibrium Density-Gradient Pgs 392
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Centrifuges
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Differential -spinning homogenate yields pellet & supernatant -increasing centrifugal force (gravity) to isolate organelles based on mass -heavy organelles, i.e., nuclei pellet at low force of gravity (600 g) 4 g force to get mitochondria & lysosomes -3x10 5 g needed for ribosomes
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Very crude “velocity” 5-20% sucrose Fig. 3-34
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-at high speed, organelles migrate to sucrose layer equal their own density Fig. 9-26 Equilibrium Density Gradient -homogenate is applied to a gradient of sucrose -separation based on density
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organelle? -good approach, but are the proteins biologically active?
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This note was uploaded on 03/04/2010 for the course SCI 2290 taught by Professor Kraj,dean,z during the Spring '10 term at UWO.

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Jan 11 - Microscopes & Proteomic Techniques...

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