3. Introduction
• Positive and negative electrical charges are frequently
associated with biomolecules. When placed in an electric
field, charged biomolecules move towards the electrode
of opposite charge due to the phenomenon of electrostatic
attraction.
• Electrophoresis is the separation of charged molecules in
an applied electric field.
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4. Types of electrophoresis:
Moving boundary electrophoresis
Zone electrophoresis
- Paper electrophoresis
- Gel electrophoresis
Based on the supporting medium there are two types of
electrophoresis.
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5. Principle:
It is type of separation technique, in this separation of
components are carried out by the migration of charged
particles under the influence of electric field with some
migration velocity.
Electrophoretic mobility ( ue ) = 6 πŋr
q
q = Charge
ŋ = Viscosity of solution
r = Radius of particle
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6. Types of gels:
Agarose gel:
For separating larger nucleic acids.
Polyacrylamide gel:
For separating smaller nucleic acids.
- SDS-PAGE - for denaturing the proteins.
Starch:
Non-denatured proteins can be separated according to
charge and size.
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7. Type Source Use Concentration
Agarose
Polysaccharide
polymer extracted
from seaweed
Separate the proteins
ranging in size from
200 kDa
0.7 % - large DNA
2 % - small
fragments
Polyacrylamide
Chemical
polymerization
reaction
Separating proteins
ranging in size from 5
to 2,000 kDa
6 %, 8 %, 10 %,
12 %
Starch Potato starch
proteins can be
separated 5-10%
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8. • Denaturing gels are run under conditions that disrupt the
natural structure of the analyte, causing it to unfold into
a linear chain.
• Denaturing conditions are necessary for proper estimation
of molecular weight of RNA.
• Urea , DMSO and glyoxal are the most often used
denaturing agents to disrupt RNA structure.
Denaturing:
• Originally, highly toxic methyl mercury hydroxide was
often used in denaturing RNA electrophoresis
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10. Preparing acryl amide gels:
Acryl amide + bisacryl amide + denaturant + buffer
Polymerization
Add butanol
• Butanol removes bubbles and makes the surface smooth.
• Gels are usually polymerized between two glass plates
in a gel caster, with a comb inserted at the top to create
the sample wells. After the gel is polymerized the comb
can be removed and the gel is ready for electrophoresis.
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11. • Buffers in gel electrophoresis are used to provide ions
that carry a current and to maintain the pH at a relatively
constant value.
• Tris/Acetate/EDTA (TAE),
• Tris/Borate/EDTA(TBE).
Buffers :
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12. The sample to analyse is
optionally mixed with a
chemical denaturant if so
desired, usually SDS for
proteins or urea for nucleic
acids.
Sampling:
Then the samples are allow to fall in to the sample wells.
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13. Instrument and working
• An electric field is applied across the gel, causing the
negatively charged proteins or nucleic acids to migrate across
the gel away from the negative electrode.
• Smaller biomolecules travel farther down the gel, while
larger ones remain closer to the point of origin.
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14. Visualization:
• DNA may be visualized using ethidium
bromide which, when intercalated into
DNA, fluorescence under ultraviolet light,
while protein may be visualized
using silver stain or Coomassie Brilliant
Blue dye.
• PAGE of rotavirus proteins stained
with Coomassie blue
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15. • After separation, an additional separation method may
be used. The gel will then be physically cut, and the
protein complexes extracted from each portion separately.
• This can provide a great deal of information about the
identities of the proteins in a complex.
Downstream processing:
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16. Applications:
• Gel electrophoresis is used in forensics, molecular
biology, genetics, microbiology and biochemistry.
• Estimation of the size of DNA molecules following
restriction enzyme digestion, e.g. in restriction mapping of
cloned DNA.
• Analysis of lipo proteins.
• Used in DNA fingerprinting.
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17. • Separation of organic acids, alkaloids, carbohydrates,
amino acids, nucleic acids.
• Used for DNA sequencing.
• Separation of serum proteins.
• Also used in antigen-antibody studies.
• Used in food industry.
• Used to study the protein mixtures.
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19. References:
•Instrumental methods of chemical analysis by B. K. Sharma;
chapter-13 ( Pg. no. C-268, 269, C-280,281 ).
•Instrumental analysis by skoog, Holler, Crouch; chapter-30
( Pg. no. 946, 947 ).
•Skoog & west fundamentals of analytical chemistry by F.
James Holler Stanley R. Crouch; chapter-34 ( Pg. no. 942 ).
•Gel electrophoresis & its applications by Pulimamidi
rabindra reddy and Nomula Raju.
• Poly acryl amide gel electrophoresis from Wikipedia, the
free encyclopedia. 19
