Vertical gel electrophoresis has several advantages over horizontal gel electrophoresis. It allows for the use of a discontinuous buffer system to separate proteins, which is not possible with horizontal gels. The technique involves pouring an acrylamide gel between glass plates to a thickness of less than 2 mm. Samples are loaded and subjected to an electric current, with cations moving toward the cathode and anions toward the anode. Proteins are separated based on their size and charge using techniques like SDS-PAGE, which involves denaturing proteins to impart a uniform charge.

1. SRIKANTH H. N.
Jr. MSc.(Agri)
Dpt. Of Plant pathology
UAS Dharwad
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2. ELECTROPHORESIS
• literally means running in the electric field
• Electrophoresis is the movement of charged particles through
an electrolyte when subjected to an electric field
• Cations move towards cathode
• Anions move towards anode
• By this technique solutes are separated by their different rates of
travel through an electric field.
• Commonly used in biological analysis, particularly in the
separations of proteins, peptides and nucleic acids
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3. ELECTROPHORESIS APPARATUS
Electrophoresis apparatus consists of
1) Buffer tank -to hold the buffer
2) Buffer
3) Electrodes
4) Power supply
5) Support media
6) Tracking dye
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4. SUPPORT MEDIA FOR ELECTROPHORESIS
1) Filter Paper
2) Cellulose acetate membrane
3) Agar or Agarose gel
4) Starch Gel
5) Polyacrylamide gel
GEL ELECTROPHORESIS
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5. GEL ELECTROPHORESIS
• Based on gel casting technique, classified in to
1) Horizontal
2) Vertical
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6. VERTICAL GEL ELECTROPHORESIS
• Slightly more complex than its horizontal
counterpart.
• Can utilizes a discontinuous buffer system,
(It is not possible to utilize discontinuous buffer
system with horizontal system).
• A thin gel (less than 2 mm) is poured between
two glass plates.
• When current is applied, a small amount of
buffer migrates through the gel from the top
chamber to the bottom chamber.
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7. VERTICLE GEL
ELECTROPHORESIS
PAGE
CONTINUOUS
PAGE
NATIVE
SDS
DIS-CONTINUOUS
PAGE
NATIVE
SDS
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8. PAGE is always vertical
Acrylamide cannot be used for horizontal
systems because
• in HGE gels are cast in a tray which is
exposed to atmospheric oxygen.
• Oxygen inhibits the polymerization of
acrylamide so it interferes with the creation
of the gel.
VERTICLE GEL
ELECTROPHORESIS PAGE
CONTINUOUS PAGE
NATIVE
SDS
DIS-CONTINUOUS
PAGE
NATIVE
SDS
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9. Continuous gel
electrophoresis
single separating gel
Discontinuous gel
electrophoresis
Two gel layers, a lower
resolving gel and an
upper stacking gel
VERTICLE GEL
ELECTROPHORESIS
PAGE
CONTINUOUS
PAGE
NATIVE
SDS
DIS-CONTINUOUS
PAGE
NATIVE
SDS
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10. Continuous gel electrophoresis
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11. Discontinuous gel electrophoresis
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12. Purpose of using two layers of gel
• Stacking gel (5 %) needed to concentrate all the
proteins in one band, so that they will start
migrating in running gel all at the same time
• separating gel (12 %) or running gel allows to
separate the proteins based on their molecular
weight
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13. PAGE
Native-PAGE-Proteins may be run in their native state
(i.e in tertiary or quaternary structure
VERTICLE GEL
ELECTROPHORESIS
PAGE
CONTINUOUS PAGE
NATIVE
SDS
DIS-CONTINUOUS
PAGE
NATIVE
SDS
SDS-PAGE- Alternatively, a chemical denaturant
added to remove this structure and turn the molecule
into simple polypeptide chain whose mobility
depends only on its length and mass-to-charge ratio.
Proteins are amphoteric
They carry positive, negative or zero net charge depending on
the pH of their local environment
Isoelectric point – pI
If pH > pI –negatively charged
If pH < pI positively charged
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14. • SDS-PAGE is the most commonly procticed GE
technique used for proteins.
• Very large proteins (subunit sizes >200 kDa)
are difficult to electrophoretically separate
on polyacrylamide gels.
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15. Outline of procedure for SDS-PAGE
Stain and image the gel
Run the gel
Voltage > 200 for 30 to 40 minutes
Load the sample
Heat the sample @ 90-95 0c along with SDS
Prepare sample
Prepare gel and assemble the electrophoresis cell
12 % running gel 5 % stacking gel
Prepare buffer
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16. Requirements for SDS-PAGE
1. Protein sample
2. Vertical electrophoresis unit
3. Buffer
4. Polyacrylamide gel
5. Sodium Dodecyl Sulphte (SDS)
6. Tracking dye
7. Stains
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17. 2) Vertical gel electrophoresis unit
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18. 3) Buffer
Buffer system include the buffers used to
• Cast the gel
• Prepare the sample (sample loading buffer)
• Fill the electrode reservoir (Running buffer)
Purpose
• Establish pH and provide ions to support conductivity
Common buffers in PAGE
1. Tris-HCL
2. Tris-glycine
3. Tris-acetate
4. Tris-tricine
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19. Tris glycine (5x)
• Tris – 15 g
• Glycine – 72 g
• SDS – 5 g
In 1 L sterile distilled water
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20. 4) Polyacrylamide Gel
• It is a polymer of acrylamide
• Acrylamide (C3H5NO) Is a chemical compound
• White odorless crystalline solid soluble in water, ethanol, ether and chloroform.
• Stable, chemically inert and electrically neutral.
• It is used to sythesise polyacrylamide
• Polyacrylamide is prepered by polymerisation of acrylamide.
• Polymerisation is initiated by ammonium persulphate (APS) with
tetramethylethylenediamine (TEMED) acting as a catalyst
• Polyacrylamide Gel It may be precast or handcast
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22. To cast Polyacrylamide gel
• Acrylamide, buffer, APS and TEMED
• Spacer plate and short plate
• Comb
• Casting frame and casting stand
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23. 5) SDS
• A strong detergent agent denature native
form of proteins and wraps around the
polypeptide backbone
• Contributes uniform negative charge to
polypeptides
• No need of this in DNA
(DNA carry negative charge with phosphate
group in both 6.8 and 8.8 pH.)
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24. 6) Tracking dye
• As proteins and nucleic acids are mostly
colourless.
• An ionic dyes of known electrophoretic
mobility are included in the PAGE sample
buffer
• Very common tracking dye – bromophenol
blue
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25. 7) Staining techniques used for
visualisation
1. Coomassie blue staining:
Stain (0.1 %) made with methanol and acetic acid
Gels are incubated in solution for 30 min.
(“G” form of dye stains only protein)
2. Ethidium bromide staining:
• Gels are incubated with aqueous solution of ethidium
bromide (0.5 µg/ml) for 5 min.
• Visualised with UV transilluminator
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26. Coomassie blue staining Ethidium bromide staining
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27. How SDS works
D:VGE Video'sHow SDS works.mp4
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28. Why vertical gel electrophoresis for
proteins….?
• Technically the porosity of the agarose gel is
higher than the SDS PAGE.
• Proteins are smaller molecules compare to
nucleic acids.
• This is the reason why we choose small porosity
(acrylamide crosslinkage) for proteins and large
porosity (agarose crosslinkage) for DNA or RNA.
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29. Can we use SDS-PAGE for seperation of
nucleic acids instead of Agarose Gel
Electrophoresis..?
• Probably you can try SDS PAGE for nucleic
acids if it's molecular weight is less than 500
Kda. and the gel percent should be 6 to 8%.
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30. Advantage of VGE over HGE
• Unlike horizontal systems, the buffer can only flow
through the gel, which allows for precise control of
voltage gradients during separation.
• When combined with the smaller pore size of the
acrylamide gel, greater separation and resolution
can be achieved with this system compared to
horizontal systems.
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