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4-423-08ProteinAnalysisStudent - Protein Analysis...

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Protein Analysis OBJECTIVES What characteristics do biomolecules, in general, and proteins, specifically, have that can be used for separation? Be able to describe the rationale and basic chemistry (not the details) behind the various purification strategies mentioned in class Be able to design a purification strategy for a mixture of proteins What differentiates preparative techniques from analytical techniques? Why is it useful to know the sequence of a protein? I Overview of Purification A. In order to study a biomolecule, you must purify it and then analyze it 1) Choose the tissue that contains the specific protein 2) Free the protein for the cell. Usually the cell must break to do so Preliminary steps: Cellular disruption - many possibilities depending on organism or tissue: a. Homogenization - with or without added abrasive b. Chemical denaturation of membranes - SDS, ethanol, etc. c. Enzymatic digestion of cell walls - e.g. lysozyme and proteinase often used to "soften" bacterial cell walls d. Ultrasonic - e. Decompression - i.e. French press 3) Purify the molecule from the rest of the stuff. B. What characteristics do biomolecules have that we could use for separation? 1) size 2) Net Charge 3) Affinity, i.e. what does the protein like and bind to? - this is correlated to function of the protein 4) Polarity? Solubility?
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C. Maintaining the protein in the process After you break the cells, the biomolecules will need the correct pH to maintain ionic character, the correct temperature so as to prevent degradative functions of lysosomes etc., and aqueous surroundings. II Overview of Characterization: Structural and/ or functional analysis A. Structural: 1. sequencing - primary structure 2. x-ray diffraction - secondary and tertiary structure B. Functional: 1. activity assay 2. localization - antibody probes III Separation on the basis of size/mass: A. Dialysis Dialysis - separates large from small molecules by diffusion across semi-permeable membrane, usually of cellulose acetate. B.Gel Filtration Size Exclusion Chromatography - also known as gel filtration. Dextran (Sephadex), agarose (Sepharose), or polyacrylamide (Bio-Gel) beads available in variety of bead sizes and pore sizes. Beads are hydrated and slurried into a column. These are beads w/pores within gel matrix into which the smaller molecules can fit. Since the smaller molecules go through the pores they take a longer time to travel through the resin bed. The larger molecules can't fit in the pores and slide down around the beads. The route for the larger molecules is shorter so they come out quicker, i.e., they elute first. Collect fractions. Proteins usually detected by uv absorbance. C. Centrifugation - can be used to separate proteins. Friction of the protein in a viscous solution will retard larger proteins more than smaller ones.
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