1.4.3 The commonly used anion exchange resin is diethylaminoethyl-cellulose DEAE-cellulose)and cation exchange resin is carboxymethyl-cellulose (CM-cellulose). 1.4.4 lon exchange chromatography may have very high resolution(specific binding)and is routinely used in protein purification. 1.4.5 New view: surface charge distribution is more crucial than the net charge (pl). pH mapping and a variety of resins(columns with different properties) should be tried
1.4.3 The commonly used anion exchange resin is diethylaminoethyl-cellulose (DEAE-cellulose) and cation exchange resin is carboxymethyl-cellulose (CM-cellulose). 1.4.4 Ion exchange chromatography may have very high resolution (specific binding) and is routinely used in protein purification. 1.4.5 New view: surface charge distribution is more crucial than the net charge (pI). pH mapping and a variety of resins (columns with different properties) should be tried
1.5 Proteins can be effectively purified by affinity chromatography. 1.5.1 This technique makes use of the binding capacity of many proteins for specific ligands chemical groups or(attached)molecules(e.g, between substrates and enzymes, antigens and antibodies, etc
1.5 Proteins can be effectively purified by affinity chromatography. 1.5.1 This technique makes use of the binding capacity of many proteins for specific ligands: chemical groups or (attached) molecules (e.g., between substrates and enzymes, antigens and antibodies, etc.)
1.5.2 The specific ligands are usually covalently cross-linked to insoluble beads 1.5.3 Specific ligand-binding proteins are retained on the column(all other nonspecific proteins are washed away from the column with low salt buffers)when a mixture of proteins(cell extract) is applied. 1.5.4 The specifically bound protein is eluted out under appropriate conditions(high concentration of ligands or salts)
1.5.2 The specific ligands are usually covalently cross-linked to insoluble beads. 1.5.3 Specific ligand-binding proteins are retained on the column (all other nonspecific proteins are washed away from the column with low salt buffers) when a mixture of proteins (cell extract) is applied. 1.5.4 The specifically bound protein is eluted out under appropriate conditions (high concentration of ligands or salts)
2. Proteins can be separated and characterized by gel electrophoresis. 2.1 Electrophoresis refers to the phenomenon that a molecule with a net charge will move in an electric field 2.1.1 The velocity of migration(v)of a molecule in an electric field depends on the electric field strength(E), the net charge(z), and the friction coefficient(f v=EZ/f
2. Proteins can be separated and characterized by gel electrophoresis. 2.1 Electrophoresis refers to the phenomenon that a molecule with a net charge will move in an electric field. 2.1.1 The velocity of migration (v) of a molecule in an electric field depends on the electric field strength (E), the net charge (z), and the friction coefficient (f): v = Ez/f
2. 1.2 The electric force Ez, driving the charged molecule toward the oppositely charged electrode, is opposed by the viscous drag (resisting force)iv, arising from the friction between the moving molecule and the medium. 2.1.3 The frictional coefficient f depends on both the mass and shape of the migrating molecule and the viscosity() of the medium(for a sphere of radius r,f=6πηr, Stoke'slaw)
2.1.2 The electric force Ez, driving the charged molecule toward the oppositely charged electrode, is opposed by the viscous drag (resisting force) fv, arising from the friction between the moving molecule and the medium. 2.1.3 The frictional coefficient f depends on both the mass and shape of the migrating molecule and the viscosity () of the medium (for a sphere of radius r, f =6r, Stoke’s law)