LS3-9-10 - Lecture 9 Protein Analysis Methods of protein...

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Methods of protein purification Methods of protein analysis Protein sequencing Protein Analysis Lecture 9 Allison p198-199 box 8.1 p269-270
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Protein Analysis Preparative techniques To purify a protein away from contaminants. Purification is measured as an increase in specific activity (mg (or enzymatic activity) of specific protein/mg total protein) Analytical techniques To analyze pure proteins or proteins in a mixture. In the latter case, little or no purification is necessary.
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Specific proteins can be purified from cell extracts Cytoplasm = everything inside cytoplasmic membrane Cytosol
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Separation of organelles by equilibrium density-gradient centrifugation Separation is based on the density of the substance, not its mass or shape
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Proteins can be separated from one another by column chromatography Partitioning proteins between two phases, liquid and solid Gel filtration chromatography (size exclusion) Ion exchange chromatography Affinity chromatography Requires an assay to follow protein, e.g. enzyme activity Analytical techniques
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Based on size of protein Large proteins are excluded from the pores and pass through the column faster. Small proteins enter the pores in the beads and are retarded in their migration. Gel Filtration (size-exclusion) Column Tool Box 8.1
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Ion-exchange chromatography Based on charge difference among proteins: Charge on proteins is sum of charges on amino acids Negatively charged beads bind positively charged proteins. e.g. phosphocellulose for DNA-
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LS3-9-10 - Lecture 9 Protein Analysis Methods of protein...

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