Electrophoresis is a method used to separate charged particles such as proteins and nucleic acids based on differences in their migration speed in an electric field. There are several types of electrophoresis including agarose gel electrophoresis, SDS-PAGE, isoelectric focusing, capillary electrophoresis, and two-dimensional electrophoresis. During electrophoresis, charged particles migrate through a gel or capillary towards an electrode with the rate of migration depending on factors like the particle's charge, size, and shape.

1. ELECTROPHORESIS:
PRINCIPLES AND TYPES
Presented by,
Sabhyata Chaubey
M.Sc. [Bt] Semester- 1st
Roll No. - 201810901010011
Under the guidance
of
Dr. Sunil Kumar
Associate Professor1

2. ELECTROPHORESIS
• Electrophoresis: method used to
separate charged particles from one
another based on differences in their
migration speed.
• Electro=Electric; phoresis= Migration;
Carry across.
• Rate of migration
– Depends on charge and size
– Separation based on differences in
charge-to-size ratios.
• High efficiency and resolution
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3. PRINCIPLE:
• Separation of charged particles based on differences in
their migration speed.
• Biological molecules exist in a solution as electrically
charged particles at a given pH.
- anionic (positively charged/basic)
- cationic (negatively charged/acidic)
• When electricity is applied to the medium containing
biological molecules, depending on their net charge and
size, they migrate differentially.
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4. Migration Depends on
• Strength of electric fields.
• Temperature
• Features of the molecule
– Net charge of molecule
– Size of molecule
– Shape of molecule
• Features of the Gel
– Gel type
– Gel concentration
• Buffer Type/pH.
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5. ELECTROPHORESIS TYPES
• Agarose gel electrophoresis
• SDS PAGE (Sodium dodecyl Sulphate Polyacrylamide
gel electrophoresis)
• Isoelectric focusing
• Capillary electrophoresis
• Two dimensional electrophoresis
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6. A. GEL ELECTROPHORESIS
• Use of a gelatinous
material.
• The gel acts as a support
medium.
• Used to separate proteins
or nucleic acids.
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7. • Starch-Rarely used
• Polyacrylamide-Protein, small nucleic
acid fragments
• Agarose-Nucleic acids, large proteins
• Cellulose acetate-Proteins
Gel Types
Commonly
used
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8. AGAROSE GEL ELECTROPHORESIS
• A technique used to separate DNA fragments according to their size.
DNA segments loaded into wells at one end of gel
Electric current is applied to the gel
Negatively charged DNA fragments move towards positive electrode
small fragments move through the gel faster than large ones.
gel is stained with a DNA-binding dye
DNA fragments can be seen as bands
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9. GENERAL REPRESENTATION OF
GEL ELECTROPHORESIS
APPARATUS
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10. GEL
ELECTROPORESI
S
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11. TECHNIQUE
Sample preparation
Gel, buffers, etc. preparation.
Load markers
Load samples
Running of the gel
Staining of the gel
Photography, gel documentation
Interpret/analysis of gel 11

12. DNA MARKER
• Ethidium Bromide
• Powerful mutagen but it works well.
• Cheap, sensitive, easy to use, fast.
• Binds to DNA .
• Fluorescence under UV lamp and visualizes of DNA on
the Gel.
• Can be added directly into the gel and/or buffer or
• Gel can be stained after run.
• Concentration 0.5-1ug/ml for staining gels.
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13. VOLTAGE
• More voltage, more quick gel runs.
• But it will lead to
– Low resolution.
– Increase temperature
• As a result, low quality separation.
• ≤5-8 V/cm of gel length.
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14. Buffer
• Provides ions in solution for electrical conductivity.
• Prevents the pH changing.
• Common using buffers:
– Tris Borate EDTA (TBE)-Stable, expensive, PAGE, long
separation time.
– Tris Acetate EDTA (TAE)-Inexpensive, short separation
time.
– Tris Phosphate EDTA (TPE)
• RNA
– Sodium phosphate Buffer
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15. B. SDS PAGE
• PAGE (Polyacrylamide Gel Electrophoresis), is an
analytical method used to separate components of a
protein mixture based on their size.
• Simple to use and highly reproducible technique.
• Provide information of the molecular weight, charged,
subunits, purity of protein mixture.
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16. • Native PAGE:
– Separates folded proteins by charge, size, and shape.
• Denaturing gel electrophoresis
– Separates folded proteins by size.
TYPES
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17. • SDS is a detergent
with a strong protein-
denaturing effect
• Binds to the protein
backbone.
• Protein may be
visualised using silver
stain or Coomassie
Brilliant Blue dye.
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18. • Before separating the proteins by mass, they are treated
with sodium dodecyl sulfate (SDS) along with other
reagents.
• This denatures the proteins and binds a number of SDS
molecules roughly proportional to the protein's length.
• Since the SDS molecules are negatively charged, the result
of this is that all of the proteins will have approximately the
same mass-to-charge ratio as each other.
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19. C. ISOELECTRIC FOCUSING
• Electrophoretic method that separates proteins according to
the iso-electric points.
• Ideal for separation of amphoteric substances.
• Separation is achieved by applying a potential difference
across a gel that contain a pH gradient.
• Requires solid support such as agarose gel and
polyacrylamide gel.
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20. • Isoelectric focusing gels contains synthetic buffers called
ampholytes that smooth the pH gradients.
• Ampholytes are complex mixtures of synthetic
polyamino-polycarboxylic acids.
• Commercially available ampholytes are-
- BIO-LYTE
- PHARMALYTE
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21. ISOELECTRIC FOCUSING
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22. D.CAPILLARY
ELECTROPHORESIS
• Capillary electrophoresis is an analytical technique
that separates ions based on their electrophoretic
mobility with the use of an applied voltage.
• Electrophoretic mobility- The rate of migration (usually
in cm/s) per unit electric field strength (usually V/cm) of
a charged particle in electrophoresis.
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23. • Electrophoretic mobility depend upon
-charge of the molecule
-viscosity
-atom's radius.
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24. E. TWO DIMENSIONAL
ELECTROPHORESIS
• Two-dimensional gel electrophoresis, abbreviated
as 2-DE or 2-D electrophoresis, is a form of gel
electrophoresis commonly used to analyze proteins.
• 2-D electrophoresis begins with electrophoresis in the
first dimension and then separates the molecules
perpendicularly from the first to create an
electropherogram in the second dimension.
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25. • In electrophoresis in the first dimension, molecules are
separated linearly according to their isoelectric point.
• In the second dimension, the molecules are then
separated at 90 degrees from the first electropherogram
according to molecular mass.
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