lecture 6 - 6-1MCB354Lecture 6Reading in Lehninger:Chapter...

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Unformatted text preview: 6-1MCB354Lecture 6Reading in Lehninger:Chapter 3Protein Purification & Characterization Overview of Protein Purification Proteins can be purified by a number of methods, usuallycolumn chromatography.Progress of the purification can be followed using apurification tableto monitor:-total protein -total activity-specific activity (total act./total prot.)Methods of Separation: differential centrifugation, solubilityand column chromatography6-2Issues to Consider in Protein Purification Goal: separate your target protein from other macromolecules and small molecules; usually for structural and functional studies Mild Conditions; avoid denaturation of target; 0 to 4 C;Avoid pH extremes. Detection MethodsMultiple Steps; often more than one purification techniqueIs required to achieve the desired level of target purity 6-3Protein Purification TutorialIntro table of common techniquesAffinity chromatographybinding to small moleculesIon exhangechromatographyIsoelectricpoint(charge)Size-exclusion chromotographySize / shapeMethodsPropertyTable of common methods of protein purificationsolubilityPrecipitation with ammonium sulfate (salting out)**Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes. It is commonly one of the first steps in a purification scheme....
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lecture 6 - 6-1MCB354Lecture 6Reading in Lehninger:Chapter...

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