ELECTROPHORETIC METHODS,Basic principles of electrophoresis,Buffers,Gel electrophoresis.Agarose gels

1. ELECTROPHORETIC METHODS

2. Basic principles of electrophoresis
1. It is the process of moving charged
biomolecules in solution by applying
an electrical field across the mixture.
2. Biomolecules moved with a speed
dependent on their charge, shape,
and size and separation occures on
the basis of molecular size.
Electrophoresis is used: for analysis and
purification of very large molecules
(proteins, nucleic acids)
for analysis of simpler charged
molecules (sugars, amino acids,
peptides, nucleotides, and simpler
ions).

3. When charged molecules are
placed in an electric field,
they migrate toward either
the positive (anode) or
negative (cathode) pole
according to their charge.
1.
2.
3.
4.
Factors influenced
electrophoresis mobility:
net charge of the
molecule
size and shape
concentration of the
molecule in solution

4. Electrophoresis is carried out by applying a thin
layer
Aqueous protein solution is immobilized in a solid hydrophilic
support.
Solid matrix with pores which are used:
• paper
• starch
• cellulose acetate
• polyacrylamide
• agar/agarose
Molecules in the sample move through porous matrix at
different velocity.

5. • Electrophoresis can be one
dimensional (i.e. one plane of
separation) or two dimensional.
• One dimensional electrophoresis is
used for most routine protein and
nucleic acid separations. Two
dimensional separation of proteins is
used for finger printing , and when
properly constructed can be
extremely accurate in resolving all of
the proteins present within a cell
(greater than 1,500).
• Most common stabilizing media are
polyacrylamide or agarose gels.

6. Buffers
• Function of buffer
1. carries the applied current
2. established the pH
3. determine the electric charge on the solute
• High ionic strength of buffer
– produce sharper band
– produce more heat
• Commonly used buffer
• Barbital buffer & Tris-EDTA for protein
• Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)

7. 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.

8. 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

9. Agarose – highly purified
polysaccharide derived from
agar (extracted from
seeweed), long sugar
polymers held together by
hydrogen and hydrophobic
bonds.
Acrylamide (CH2=CH-CONH2)
Polyacrylamide gel structure
held together by covalent
cross-links

10. 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
O

11. Polyacrylamide gels
CH2=CHCONH2
Acrylamide
+
CH2(NHCOHC=CH2)2
N,N,N,N-methylenebisacrylamide
Free radical catalyst
-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

12. Capillary electrophoresis
Capillaries are typically of 50 µm 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.