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2.
3.
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Factors influenced
electrophoresis mobility:
net charge of the
molecule
size and shape
concentration of the
molecule in solution
Buffers
Function of buffer
1. carries the applied current
2. established the pH
3. determine the electric charge on the solute
Gel electrophoresis
Gel is a colloid in a solid form (99% is water).
Gel material acts as a "molecular sieve.
During electrophoresis, macromolecules are forced to
move through the pores when the electrical current is
applied.
Support media
Agarose and polyacrylamide gels are acrosslinked, spongelike structure
It is important that the support media is
electrically neutral. Presence of charge group
may cause:
-Migration retardation
-The flow of water toward one or the other electrode so
called Electroendosmosis (EEO), which decrease
resolution of the separation
Agarose gels
For the separation of (1) large protein or protein
complex (2) polynucleotide 50-30,000 base-pairs
The pore size is determined by adjusting the
concentration of agarose in a gel (normally in the rank
of 0.4-4%
OH
O
CH2OH O
OH
O
O
OH
O
Polyacrylamide gels
CH2=CHCONH2
Acrylamide
CH2(NHCOHC=CH2)2
N,N,N,N-methylenebisacrylamide
Capillary electrophoresis
Capillaries are typically of 50 m inner diameter and 0.5 to 1 m in
length.
Due to electroosmotic flow, all sample components migrate towards
the negative electrode.
The capillary can also be filled with a gel, which eliminates the
electroosmotic flow. Separation is accomplished as in conventional gel
electrophoresis but the capillary allows higher resolution, greater
sensitivity, and on-line detection.