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Dr. Kim OH for Exam II

Dr. Kim OH for Exam II - Professor Kim's OH Monday December...

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Professor Kim’s OH – Monday, December 3, 2007 Enzyme, protein assay = fundamental Crude Extract Preparation Purpose: lysis and enzyme collection into buffer (crude homogenate) Important technical points: using a blender, time (bursts—in order to reduce thermal energy), lower oxidation, decrease protease activity (keep on ice as much as possible). Enzyme assay degradation Protein hydrophilic and hydrophobic regions are still intact so it doesn’t affect binding to the dye Two major procedures involved in this crude extract step Centrifugation and Filtration Purpose: removing insoluble debris (lipids, intact membranes, organelles) that is less dense (through cheesecloth filter). Centrifugation divides the components into soluble (supernatant) and insoluble (pellet) portions. Force of buoyancy, force of centrifugation (don’t need to know the equations). But need to know that more dense than the solvent volume below will be displaced If you mess up on any of this stuff, will this affect your percent yield? No, you will not be able to get valid measurements because of all the junk in your crude homogenate. Junk interferes with dye-binding assay. Interferes with protein assay but not enzyme assay. Ammonium Sulfate Precipitation Separating protein Characteristic that this procedure is using: solubility Ability to interact with the solvent Changing solubility properties in the solution and precipitate out the target Ammonium sulfate against macromolecules inside the crude extract. Ammonium sulfate interacts more strongly with water than any of the other macromolecules b/c smaller SA and are charged ions. Ability to attract water is much stronger. Ammonium sulfate can remain soluble at colder temperatures (not imp) How does this kind of addition alter proteins’ ability to remain solute? Taking water away increase the likelihood that macromolecules can interact directly with each other. Reorientating them to the salt, not to the protein. Creating water shells that point to a certain type of polarity. Protein (amine group on protein) Electrostatic repulsion, destabilizing ____? Collision of macromolecules are very random. Decrease surface area of macromolecules to water. Create densely packed macromolecules. Reorients, repulsion drives collision of macromolecules random interactions, expose dense bodies (may be asked to apply this concept) Major technical points : thermal energy, adding salt slowly so you don’t have immediate
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increase in saturation of microenvironment ( Any proteins in that microenvironment will become aggregates and lost to the pellet, giving rise to higher protein amounts ). Stir slowly. “Vigorous stirring causes more oxidation and surface denaturation, which will disrupt enzyme structure and activity.” What is percent saturation (make sure you know what that is)?
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