L2 - READING FOR THURSDAY Ch. 3 125-142; 153-159; Ch. 10...

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Unformatted text preview: READING FOR THURSDAY Ch. 3 125-142; 153-159; Ch. 10 ALL We are getting the TA ofce hours together Please see the web for details First problem set is available on the web Podcasting is happening so last weeks lecture is on the web at media services Some Techniques in Cell Biology Making extracts Protein fractionation by size, Cell culture to test fractions Visual cells by phase contrast PCR to identify archaea FACS to purify archaea More chromatography, example of ion exchange, to purify active protein Structural analysis by X-ray crystallography to get info Chemical biology to synthesize similar small molecules that could act the same way. . Suppose you are a marine biologist that searches for new natural products that could be useful anti-cancer drugs. You are diving at a coral reef off of Tahiti, and you notice an unusual sponge that you have never seen before. You collect a sample, take it back to your lab, make an extract, and find that it contains a compound that kills cancer cells, but not normal cells. You are very excited and want to figure out where this compound comes from. Because you are a good marine biologist, you suspect that the compound actually comes from a micro-organism that has a symbiotic relationship with the sponge. You examine the sponge under a microscope and confirm that there are micro- organisms that are part of the sponge. You want to purify this cancer fighting compound. How do you do this? Phil Crews and his team http://chemistry.ucsc.edu/mnpr/expeditions.htm 1) Had a marvelous working vacation in Tahiti. 2) You collect a sample, take it back to your lab, make an extract and separate proteins based on size. Tools in cell biological research- Detergents and membrane solubilization Detergents Membrane solubilization with detergents Triton X-100 (a non-ionic detergent) dissolves membranes and solubilizes membrane proteins without affecting their structure/ function. SDS (an ionic detergent) dissolves membranes and denatures protein structure. You can also break open cells by osmotic shock, ultrasonic vibration or a blender + + The centrifuge is a key biochemical tool If you want to fractionate a cell extract, you centrifuge at different velocities. If you want to isolate a cell, its nuclei or cytoskeleton, you do a low speed spin Cell Fractionation Low speed pellet (1,000 x g) 10 minutes If you want to fractionate a cell to isolate an organelle, you do a medium speed spin Cell Fractionation (20,000 x g) 20 minutes Medium speed pellet If you want to fractionate a cell to isolate smaller organelles, you do a high speed spin. Cell Fractionation (80,000 x g) 1 H High speed pellet If you want to fractionate a cell to isolate a macromolecule, you do a very high speed spin Cell Fractionation (150,000 x g) 3 H VERY High speed Finer degrees of separation are achieved by centrifuging through a salt/sucrose gradient Differential velocity sedimentation Velocity sedimentation: separates based on size and shape equilibrium centrifugation: fractionates based on buoyant density 1) Size: gel...
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This note was uploaded on 03/28/2010 for the course MCD BIO 110 taught by Professor Hinck during the Fall '09 term at UCSC.

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L2 - READING FOR THURSDAY Ch. 3 125-142; 153-159; Ch. 10...

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