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Cell Disruption (Lysis) release proteins while preserving their…

- - - - Protein Concentration
        
        Chromatographic
        Separation
        
        Analysis & Validation
        
        Storage
        
        -80°C
        
        stabilizers
        
        ANALYTICAL METHODS
        analyze the protein
        examine the behavior of mixture components in order to gain insight into their:
        
        nature
        
        structure
        
        physicochemical properties
        
        protein electrophoresis
        
        blot transfer
        
        immuno-staining
        
        gel imaging
        
        SDS-PAGE
        
        western blot
        
        activity assays
        
        type
        
        ≠equilibria beetween the 2 phases
        
        separation occurs becuse molecule establishes these equilibria differently based on size, charge, polarity, binding ability
        
        molecular exclusion/size-based equilibrium
        
        liquid inside porous beads + mobile liquid phase
        
        separation based on size
        
        large molecule excluded from pores and move faster
        
        small ones enter pores and are delayed
        
        Size exclusion chromatography (SEC)
        
        Molecular exclusion chromatography
        
        gel filtration chromatography
        
        Affinity equilibrium
        
        immbilized ligand (on a solid support) and a mobile liqid phase
        
        separation is based on specific biological interactions --> binding affinity
        
        only molecules with high affinity bind strongly and are retained
        
        dye-ligand chromatography
        
        affinity chromatography
        
        the beads in the column have a covalently attached chemical group called ligand
        
        protein mixture added --> any proteins with affinity for this ligand bind to the beads --> migration to the matrix retarded
        
        Ion exchange equilibrium
        
        solid stationary ion exchanger + mobile aqueous electrolyte phase
        
        separating according to charge
        
        charged solute interact electrostatically with oppositely charged groups on the stationary phase
        (a positive charged prot. bind with a negative bind prot.)
        
        Ion exchange chromatography
        
        affinity of each protein for the charged groups --> affected by the pH and the concentration of competing free salt ions in the surrounding solution
        
        the column matrix is a synthetic polymer (resin):
        
        anion exchangers
        
        1 more item...
        
        cation exchangers
        
        1 more item...
        
        adsorption equilibrium
        
        solid stat. p. + liquid mobile p.
        
        solute mol. bind to the surface of the solid phase with different strengths
        
        separation depends on how strongly each compound interacts with the surface
        
        Hydrophobic interaction chromatography
        
        adsorption chromatography
        
        partition/distribution equilibrium
        
        liquid stationary p. + liquid mobile p.
        
        solute distributes itself between two immiscible liquid phases
        
        based on differences in sulubility and partition coeficient
        
        Partition chromatography
        
        Reverse-phase chromatography
        
        components of a mixture are separated based on their distribution in:
        
        stationary phase
        
        solid or liquid
        
        attached toan inert matrix
        
        packed in a glass or metal column
        
        remains fixed
        
        different components to separate as they interact differently with each phase
        
        one must be chosen
        
        1 more item...
        
        mobile phase
        
        liquid or gas
        
        moves through the stationary phase by gravity or via a pumpin system
        
        Ultrafiltration
        
        pore size: 0.01-0.1 microm
        
        Precipitation
      - Dialysis
      - Desalting columns
    - - Salting in
        
        low salt & low ionic strength
        
        protein more soluble
      - Salting out
        
        high salt & low ionic strengh
        
        proteins precipitate
    - - isoelectric point --> net charge =0
        
        tends to precipitate
  - - - low-speed centrifugation, pellet contains:
        
        whole cells
        
        nuclei
        
        cytoskeletons
        plasma membranes
        
        supernantant subjected to medium-speed centrifugation:
        
        mithocondria
        
        lysosomes
        -peroxisomes
        
        supernant subjected to high-speed centrifugation:
        
        microsomes (fragment of ER)
        
        small vesicles
        
        supernant subjected to very high-speed centrifugation:
        
        ribosomes
        
        large macromolecules
  - - - negative pressure
    - - positive pressure
    - - microfiltartion:
        pore size: 0.1-10 microm
- - - - adding a few or many amino acid residues to one or both ends