Biomedical Engineering Reference
In-Depth Information
Electrophoresis
Electrophoresis is a method used to separate macromolecules from
complex mixtures by application of an electric field. The macromolecules
are placed at one end of a matrix (referred to as a “gel”) and are then sub-
jected to the electrical current. Different macromolecules in the mixture
will migrate at different speeds, depending on the nature of the gel and
the characteristics of the macromolecules. Electrophoretic techniques
can be applied to the separation of any macromolecule, including nucleic
acids (DNA and RNA), proteins and carbohydrates. However, the princi-
ples of the separation and the matrices used may differ depending on the
molecules that need to be separated. The electrophoretic separation of
macromolecules in gels gives superior resolution to gradient separation
techniques. The development of electrophoretic separation techniques
was a major advance in the ability to resolve and characterize proteins
and nucleic acids. For his efforts in development of electrophoresis as a
method to separate proteins from complex mixtures, Arne Tiselius won
the Nobel Prize in Chemistry in 1948.
Polyacrylamide gel electrophoresis (PAGE)
The use of PAGE gels have had a major impact on our ability to
resolve proteins in complex mixtures, estimate their size, and deter-
mine some of their properties, it is one of the standard methods in
biomedicine, in part because PAGE geis are so versatile. The devel-
opment of PAGE gels, particular denaturing PAGE gels, has its roots
in protein biochemistry. Two of the early pioneers in the development
of modern electrophoretic separation techniques, F.W. Studier and J.V
Maizel, Jr., recently recounted the development of slab gels and SDS-
PAGE technology in brief reviews in Trends in Biochemical Sciences
(3,4).
Polyacrylamide gels are formed by the chemical cross-linking of
acrylamide (the chemical monomer) with the chemical agent N,N'-
methylene bisacrylamide (typically called “bis-acrylamide”). The poly-
merization reaction proceeds as a free-radical catalysis and is
initiated by ammonium persulphate and the base TEMED (N,N,N',N'-
tetramethylenediamine). More chemistry than you wanted? The bottom
line: PAGE gels are remarkably versatile for performing high resolution
separation of proteins and nucleic acids (such as for resolving DNA
ladders in DNA sequencing reactions, or for RNase protection assays).
In the case of proteins, PAGE gels can be used to separate them ac-
cording to their size or charge, depending on other features of the gel
matrix.
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