lec 28 - Lecture 28 Purification detection and...

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Unformatted text preview: Lecture 28: Purification, detection and characterization of proteins I (Ch. 3; p. 86-93) Protein purification Physical and chemical criteria: Mass (and shape) Density Charge Binding affinity Centrifugation Electrophoresis Chromatography Centrifugation: mass/density Differential centrifugation: Separation according to mass (= size) Separation into pellet and supernatant Can be repeated at different speeds and times to separate sequentially various organelles/molecules Centrifugation: Sedimentation unit = S (svedberg) Ex.: ribosome subunits 40S, 60S Differential centrifugation Examples of typical conditions for sedimentation (approximate, see above for parameters): Nuclei: Low speed centrifugation (~1000 g) for 5-10 min. Mitochondria: Medium speed centrifugation (~10-20.000 g) for 20-30 min Small secretory vesicles, ribosomes: High speed centrifugation (~50-150.000 g) for 15-60 min Protein complexes: High speed centrifugation (~ 100-200.000g) for > 1 hour Proteins: Ultra-high speed centrifugation (>100.000) for several hours Sedimentation depends on acceleration (g) and time length of centrifugation Acceleration is function of: Speed (rpm = revolutions/min) R = Radius (Size of rotor, length of the tube) Thus results of centrifugation depend on type of rotor, type of tube, speed and length of centrifugation Sedimentation of particles/molecules/organelles also depends on mass: for a given mass, acceleration must be high enough to win over thermal (Brownian) movements, which otherwise will „mix‟ particles again and prevent sedimentation. Example: Take 3 tubes with water and add various „particles‟, shake and let stand vertical on the bench. Acceleration = 1g. A green pea, even if tube is well shaken to „mix‟, will fall to the bottom within a fraction of...
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This note was uploaded on 12/07/2010 for the course BIO BIOL 200 taught by Professor Frogatto during the Fall '10 term at McGill.

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lec 28 - Lecture 28 Purification detection and...

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