ELECTROPHORESIS

2.  The movement of charged particles (ions) in
an electric field resulting in their migration
towards the oppositely charged electrode is
known as electrophoresis.
 Used for the separation of plasma proteins,
lipoproteins & immunoglobulins.

4.  The rate of migration of ions in an electric
field depends on several factors that include
shape, size, net charge and solvation of the
ions, viscosity of the solution & magnitude of
the current employed

5.  In this technique, the sample is applied on a
strip of filter paper wetted with desired
buffer solution.
 The ends of the strip are dipped into the
buffer reservoirs in which the electrodes are
placed.
 The electric current is applied allowing the
molecules to migrate for sufficient time.

6.  Paper is removed, dried & stained with dye.
 The colored spots can be identified by
comparing with a set of standards run
simultaneously.
 For serum proteins, whatman No.1 filter
paper, tris buffer pH8.6 & stains amido black
bromophenol blue are used.
 Serum proteins are separated into five bands-
albumin, α1, α2, β & γ- globulins.

7.  It involves the separation of molecules based
on their size, in addition to the electrical
charge.
 The large molecules move slowly in this.
 The resolution is much higher in this
technique.
 Serum proteins can be separated to about 15
bands.

8.  Commonly used gel are agarose &
polyacrylamide, sodium dodecyl sulfate (SDS).
 Polyacrylamide is employed for the
determination of molecular weights of
proteins – known as SDS-PAGE.

9.  It is based on the immobilization of the
molecules at isoelectric pH.
 Stable pH gradients are set up covering the
pH range to include the isoelectric points of
the components in a mixture.
 As the electrophoresis occurs, the molecules
(say proteins) migrate to positions
corresponding to their isoelectric points, get
immobilized & form sharp stationary bonds.

10.  The gel blocks can be stained & identified.
 By isoelectric focussing, serum proteins can
be separated to as many as 40 bands.
 lsoelectric focussing can be conveniently
used for the purification of proteins.

11.  This technique involves combination of the
principles of electrophoresis & immunological
reactions.
 lmmunoelectrophoresis is useful for the
analysis of complex mixtures of antigens &
antibodies.

12.  Textbook of Biochemistry – U Satyanarayana