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Chemistry 1027A/B
Terry Biggs

Biochemistry 2280 Protein PurificationLecture 5 Protein purificationWhy purify1 For medical useEx Pure antibodies are required for chemotherapy and autoimmune disorders2 To study structure and functionDifficult to understand activity in mixturesMust be pure to determine structure3 For use as a reagent in the laboratoryEx Purified enzymes are essential for manipulating DNAThe Purification processProteins are present in cells or secretions and are thus complex mixturesusually many different proteinsPlus otherDNA lipid small molecules etc contaminants which must be removedProteins may differe in many propertiesSize and shapeChargeLocationSurface hydrophobicityNeed an assay methodMust be able to easily detect the protein of interest in a mixture Must be able to measure some property of the protein of interestDecide on a starting materialHuman tissues plants bacteria etcPreparative vs Analytical methodsStart w preparative then proceed w analyticalPreparative methods homogenize break up materialLarge scalemilligrams to kilogramsDivide mixture into fractionsAnalytical methods detect specific protein of interestSmall scalemicrograms or lessSmall samples of fractions for analysisPreparative methods Step 1HomogenizeFollowing are techniques to break open tissue or cells to release proteinsSonication expose to high frequency sound wavesGrind it upBlendershomogenizersPressure Ex A way to break open yeast cells is to put them under extreme amt of pressureDetergents Compatible buffer solution is requiredBuffer prevents huge fluctuations of pH which keeps the protein in its native stateThe result is homogenateContains largesmall molecules from the cytosol and all of the membraneenclosed organellesPreparative methods Step 2Centrifugation
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