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Protein Separation Techniques

Proteins can be seperated by Gel Electrophoresis and Displayed

A molecule with a net charge will move in an electric field.  This phenomenon, termed electrophoresis offers a powerful means of separating proteins and other macromolecules such as DNA and RNA.  The velocity of migration of a protein (or any molecule) in an electric field depends on the electric field strength, the net charge on the protein and the frictional coefficient.

The electric force driving the charged molecule toward the oppositely charged electrode is opposed by the vicious drag arising from friction between the moving molecule and the medium.  The frictional coefficient depends on both the mass and shape of the migrating molecule and the viscosity of the medium.

Electrophoresis separation is nearly always carried out in gels (or in solid supports such as paper) rather than in free solution for two reasons.  First gels suppress connective currents produced by small temperature gradients, a requirement for effective separation.  Second gels serve as molecular sieves that enhance separation.  Molecules that are small compared with the pores in the gel readily move through the gel, whereas molecules much larger than the pores are almost immobile.  Intermediate-size molecules move through the gel with various degrees of facility.  Polyacrylamide gels are choice supporting media for elelectrophoresis because they are chemically inert and are readily formed by the polymerization of acrylamide.  Moreover, their pore sizes can be controlled by choosing various concentrations of acrylamide and methylenebisacrylamide (a cross-linking reagent) at the time of polymerization.


 

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