Ch3 - Chapter 3 Techniques for Characterizing Proteins l...

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Chapter 3 Techniques for Characterizing Proteins l Amino acid sequence l 3D Structure of Proteins l Catalytic activity l Binding to other molecules All these require purification of protein “In vivo” (in the cell) antibodies and fluorescent probes monitor protein function genome proteom e
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Steps in Protein Purification Grow cells which produce protein (engineered bacteria or insect cell culture) Release from cells Separate from other proteins (and nucleic acids, lipids) :MW, charge, hydrophobicity, affinities Prep Analyze physical properties of purified protein Assay activity of protein (monitor A340 for NADH) Each Protein requires its own purification scheme
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Fractionation Schemes for Separating Components of Cells are Developed by Trial and Error Harvest and break cells (homogenation) Centrifugation separates by density Salting out: precipitation of proteins affects centrifugation 100 mM NH4SO4: SN 200 mM: pellet
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Dialysis Provides Crude Separation by Molecular Weight Dialysis Tubing is Designed with a Discrete Molecular Weight Cutoff: (MWCO)
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Gel Filtration Chromatography  Separates by MW Types of Beads: Sephadex Sepharose Biogel: Beads of Different Sizes
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Anion Exchange Chromatography Cation- Exchange: For separating +charged proteins Anion-Exchange: For separating – charged proteins
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Affinity  Chromatography Separate proteins by their preference for a binding partner: e. g. To purify a nucleic acid binding protein, put nucleic acid in your column Engineer bacteria to produce your protein attached to: maltose binding protein, Glutathione binding protein, histidines (his-tag + nickel column) HPLC
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Analysis of Protein Purity Gel Electrophoresis v=Ez/f F=6 πη r Compact (small?) molecules move fastest SDS-polyacrylamide (denaturing) gels Acrylamide cross-linked with bis- acrylamide to form a gel SDS attaches negative charge to protein (~1/2 aa acids) Mercaptoethanol reduces disulfides
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or electrophoresis “Size markers” Separation of proteins in an SDS_acrylamide gel Can visualize 0.1 g (coomassie μ blue) or 0.02 g (silver stain) μ
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Isoelectric Focusing: pH gradient in an electric field separates proteins by number of acidic/basic groups In 2-D protein electrophoresis, resolution is improved by separating proteins according to both MW and charge/pH
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Ch3 - Chapter 3 Techniques for Characterizing Proteins l...

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