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Simple notes on electrophoresis
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Simple understanding on the topic electrophoresis
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ELECTROPHORESIS Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein
molecules based on their size and electrical charge.
An electric current is used to move molecules to be separated through a gel.
Pores in the gel work like a sieve, allowing smaller molecules to move faster than
larger molecules.
The conditions used during electrophoresis can be adjusted to separate molecules in
a desired size range.
Principle
When charged molecules are placed in an electric field, they migrate toward either
the positive or negative pole according to their charge.
In contrast to proteins, which can have either a net positive or net negative charge,
nucleic acids have a consistent negative charge imparted by their phosphate
backbone, and migrate toward the anode.
Factors affecting on Electrophoresis:
The rate of migration (Separation of particles) during electrophoresis will depend on the
following factors:
1. The Sample
2. The Electric Field
3. The Medium
4. The Buffer
1. The Sample:
Charge/mass ratio of the sample dictates its electrophoretic mobility. The mass
consists of not only the size (molecular weight) but also the shape of the molecule.
a)Charge: The higher the charge, greater is the electrophoretic mobility. The
charge is dependent on pH of the medium.
b) Size: The bigger molecules have a small electrophoretic mobility compared to the
smaller particles.
c) Shape: The globular protein will migrate faster than the fibrous protein
, 2. The Electric Field:
The rate of migration under unit potential gradient is referred to as “Mobility of the
ion”. An increase in potential gradient increases the rate of migration.
3. The Medium:
The inert medium can exert adsorption & molecular sieving effects on the
particle, influencing its rate of migration.
4. The Buffer: The buffer can affect the electrophoretic mobility of the sample in
various ways.
a)Composition:
The choice of buffer depends upon the type of sample being electrophoresed.
b) Ionic Strength:
c) pH:
The pH determines the degree of ionization of organic compounds; it can also affect
the rate of migration of these compounds. When increase pH, increases ionization of
organic acids. Decrease in pH, increases ionization of organic bases. E.g.: an
Ampholyte (Amino acid) - The amino acid has both acidic & basic properties
1.AGAROSE ELECTROPHORESIS
Principle: Gel electrophoresis separates DNA fragments by size in a solid
support medium such as an agarose gel.
Sample (DNA) are pipetted into the sample wells, followed by the application
of an electric current at the anodal, negative end which causes the
negatively-charged DNA to migrate (electrophorese) towards the bottom
(cathodal, positive) end.
The rate of migration is proportional to size: smaller fragments move more
quickly, and wind up at the bottom of the gel.
DNA is visualized by including in the gel an intercalating dye, ethidium
bromide. DNA fragments take up the dye as they migrate through the gel.
Illumination with ultraviolet lightc auses the intercalated dye to fluoresce.
The larger fragments fluoresce more intensely.
A “ladder” set of DNA fragments of known size can be run simultaneously
and used to estimate the sizes of the other unknown fragments.
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