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Lecture 28 - L ecture 28 Pu r ification Detection and...

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Lecture 28 Purification, Detection and Characterization of Proteins Protein Purification Goal: Understand the function of proteins When you want to study a molecule the simplest way to do it is to isolate the molecule: You can do this by purifying the mix Need to determine what criteria to use when purifying Physical and chemical criteria to distinguish DNA from proteins: Mass (and shape) Density Charge Binding affinity Three main methods: Centrifugation Electrophoresis Chromatography Doesn’t necessarily tell you all the criteria being used Page | 1
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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 Most useful application : Eliminate precipitates If you have an old sample and denature antibodies, you can spin for a few minutes in a normal centrifuge and take the supernatant (good antibodies) Can also be done to isolate samples 2 fractions in centrifugation: Pellet: What reached the bottom Page | 2
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Supernatant: What didn't reach the bottom Sedimentation unit: S (svedberg) Ribosome subunits are 40S and 60S Viscosity can effect centrifugation: Induces resistance on object falling down Starts to affect the shape Method: 1. Take a sample and centrifuge it so you have gravity exerted on it Using a homogonous medium Modify G according to mass or density 1. Particles settle according to mass Bigger things will tend to fall faster (more dense or bigger) 1. Stop centrifugation Decant liquid into container Example : Put water and gravel into a container Gravel will fall down and water will stay up 1G differential centrifugation Page | 3
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Rate-Zonal Centrifugation Page | 4
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Separation according to mass (and shape) Takes place through increasing dense medium (sucrose medium) Various organelles/molecules separate into bands Criteria: (1) Eventually the limit of centrifugation will be density (2) How fast the sample gets to this location Spin for limited amount of time: Small ones might not catch up to the big ones even if they have the same density Complicated mixture in tube Depend on density and on size Longer centrifugation: Equilibrium density-gradient centrifugation (according to density) Proteins will all reach the same density but organelles/membranes have different densities Great way to purify organelles with different densities Page | 5
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Method: 1. Prepare a density gradient (ex: sucrose medium) Dense at the bottom Light at the top The sample is layered on top of the gradient 1. Centrifuge: Particles settle according to mass Something that is big will fall fast until it reaches a medium with the same density It cannot go lower than that 1.
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