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Factors influenced
electrophoresis mobility:
net charge of the molecule
size and shape
concentration of the
molecule in solution
Proton
donor
Proton
acceptor
Izoelectric point
Dipolar ion
Fully protonated
form at wery low pH
Proton
donor
Proton
acceptor
Buffers
Function of buffer
1. carries the applied current
2. established the pH
3. determine the electric charge on the solute
Zone electrophoresis
Electrophoresis
Densitometer tracing
density of zones is proportional
to the amount of protein
Hypogamaglobulinemia
Normal serum
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 across-linked, 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
CH2OH O
O
OH
O
O
OH
O
Polyacrylamide gels
CH2=CHCONH2
Acrylamide
CH2(NHCOHC=CH2)2
N,N,N,N-methylenebisacrylamide
-CH2-CH-CH2-CH-CH2-CHCO CO CO
NH NH2 NH
n
CH2
CH2
NH NH2 NH
CO CO CO
-CH2-CH-CH2-CH-CH2-CHn
Detection limit
Ponceau S
1-2 g
Amido Black
1-2 g
Coomassie Blue
1.5 g
India Ink
100 ng
Silver stain
Colloidal gold
10 ng
3 ng
Isoelectric focusation
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.
Electroosmotic flow
The surface of the silicate glass capillary contains negatively-charged
functional groups that attract positively-charged counterions. The
positively-charged ions migrate towards the negative electrode and
carry solvent molecules in the same direction. This overall solvent
movement is called electroosmotic flow. During a separation,
uncharged molecules move at the same velocity as the electroosmotic
flow (with very little separation). Positively-charged ions move faster
and negatively-charged ions move slower.