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Unformatted text preview: 1 CHEM 153L Exam II Review Summary of all Topics PuriFcation and Characterization Lyse cells: Collect Extract Under Physiological conditions Reduce Thermal Energy to prevent Oxidation and Proteolytic digestion degradation Reduce air exposure surface air denaturation Remove Non Protein Macromolecules 4 Types: Protein, ats/lipids, Sugars, Nucleic Acids Isolate to a speciFc characteristic of the protein Solubility, Charge, MW, Binding to Substrates Characterize the Enzyme: Kinetics & MW F B = m s ! 2 x F f = v f F c = m m ! 2 x m m m s F c > F B, then particle moves . Centrifugation of Crude Aggregates with More Nonpolar interactions are more dense. Membranes, organelles orce of centrifugation is not sufFcient for proteins Insoluble debris low density. Supernatant (sup) Pellet Ammonium Sulfate Explain the effect of salt on solubility of molecules. Induce Aggregate formation & density increases Centrifuge to Pellet 2 Two Step Ammonium Sulfate 1 st step removes low soluble contaminants. 2 nd step removes high soluble contaminants. Can use to concentrate the sample. Reasons for Salting Out Narrow pool of biomolecules down to Proteins General clean-up to use other procedures Ex) AfFnity column: No clogging and increase binding afFnity Solution exposed to higher % saturation for a long period of time. Aggregates need thermal energy to break apart. Still part of ppt. even after salt completely dissolves. Ammonium Sulfate Survey Needed to determine range of ppt. of target enzyme/protein. If purpose is to remove other macromolecules, then objective is to generally obtain more yield than really increase purity....
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