BME 210 Lecture 12 Cells and Biomolecules Seperation

BME 210 Lecture 12 Cells and Biomolecules Seperation - 12....

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12. Cell and biomolecules separation
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Enzymatic tissue digestion Cell sorting Tissue homogenization (breaking cells open) Centrifugation Chromatography Electrophoresis Centrifugation Separation steps Biomolecules Cells
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Centrifugation Centrifugation – separation of particles and molecules using centrifugal force in a spinning tube Centrifuge Pellet
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Centrifugation factors Particle movement : m p a = F c – (F A + F f ) F c = m p ω 2 r F A = m o ω 2 r F f = fv a = 0 (v = const) F c = F A + F f v = ω 2 rV p p – ρ o )/f m p – particle mass a – particle acceleration F c - Centrifugal force F A - Archimedean (buoyant) force F f - Friction force; f – friction coefficient v – particle velocity ω – angular rotation velocity (rad/s) m o – mass of solution displaced by the particle ρ p , V p - particle density and volume ρ o – solution density F c F A F f ω r v m p Rotation measurement: • Rotation speed ω (or N,rpm) or • Relative centrifugal acceleration (ω 2 r/g - number of g )
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v = ω 2 rV p p – ρ o )/f Sedimentation speed (v) depends on: particle density and solution density (ρ p and ρ o ) particle size and shape (define volume V, friction coefficient f) • fluid viscosity (defines f) • rotation speed ω Centrifugation factors
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Centrifugation types • Both insoluble particles (cells, organelles, viruses) and soluble molecules (proteins and nucleic acids) can be separated • Two main versions: 1. Differential centrifugation 2. Gradient density centrifugation
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Differential centrifugation • A technique used to purify a specific target (e.g., cell or organelle) from a tissue homogenate • Involves stepwise increases in the speed and time of centrifugation • At each step, more dense particles are separated from less dense particles • Provides only partial purification
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• Sedimentation using a sucrose gradient , which is prepared in advance • Sucrose concentration is increasing from ≈15% at the top to ≈35% at the bottom density gradient • Density of the sample particles must be larger than that the of solution on top • Sampled mixture is placed on top. During centrifugation, different particles move at
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This note was uploaded on 04/28/2010 for the course BME eng. biolo taught by Professor Fast during the Spring '10 term at University of Alabama at Birmingham.

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BME 210 Lecture 12 Cells and Biomolecules Seperation - 12....

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