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Isolation of Proteins

1. Cell Lysis - Methods

   Waring blender Alumina grinding Sonication

   Hughes Press Lysozyme

   Detergents Freeze/Thaw

2. Differential Centrifugation
   1,000 - 2,000 xg unbroken cells and large fragments
   5,000 - 10,000 xg mitochondria
   100,000 xg for an hour ribosomes
   Supernate small molecules

3. Isolation (Things to watch out for: temperature, pH, organic solvents, denaturization...)
   1. Solubility characteristics - general classification
      1. Albumins - s. water; s saline
      2. Globulins - ss water; ss saline
      3. Prolamines - s aqueous EtOH; i water, i ethanol
      4. Glutelins - s acid; s base
      5. Scleroproteins - i aqueous systems

   2. Precipitation
      1. Salting out - usually with ammonium sulfate
      2. Alcohol precipitation
      3. Acetone precipitation
      4. Heavy metal precipitates
      5. Complexes due to charge effects
         1. Electrophoresis
         2. Adsorption and elution from gels
            1. Bulk elution
            2. Gradient elutions
         3. Ionic Exchange Resins
            1. Cationic exchanges by anionic resins
            2. Anionic exchanges by cationic resins
      6. Crystallization

   3. Criteria of Purity of your Isolated Protein
      1. Crystallinity
      2. Solubility curves - Gibbs' Phase Rule
      3. Ultracentrifugation
         1. Sedimentation rates (Svedberg units) (dynamic; non-equilibrium)
         2. Isopycnography (static; equilibrium)
         3. Diffusability (Svedberg and macro-molecular weights)
      4. Chromatographic fractionation
      5. Electrophoretic studies (various) (see Tiselius, page 2)
      6. Functional tests
      7. Molecular weight determinations
      8. Analytical data (constituents)