This lecture is about Gel Electrophoresis and a little brief about it, which is presented by Tuba Nafees she is MSc graduate in Biotechnology from University of Karachi, Sindh Pakistan. For youtube :https://www.youtube.com/watch?v=G4dwvDkxKN4

1. ESSENTIALS OF GEL
ELECTROPHORESIS
Presenter
TUBA NAFEES
M.Sc. In Biotechnology, University of Karachi
1

2. GEL ELECTROPHORESIS
PRINCIPLE
Biomolecules are separated and visualized on a gel
as they migrate through it in the presence of an
electric field.
2

3. TYPES OF GEL
• It is a purified form of agar ( Agar---
agarose+ agaropectin)
Agarose is a linear polymer repeating
units of Agarobiose (D-galactose and
3,6-anhydro-L-galactopyranose).
POLYACRYLAMIDE
• It is a polymer formed from
acrylamide subunits, crosslinked to
N,N'methylenebisacrylamide.
AGAROSE
ACRYLAMIDEBISACRYLAMIDE
D-GALACTOSE
3,6-anhydro-L-
galactose
POLYACRYLAMIDEAGAROSE
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4. AGAROSE
• Runs horizontally.
• Sets as it cools.
• Porosity is dictated by agarose
concentration ( 0.5-2 %).
• DNA fragments of 20-20,000 bp
in size
• Gel is thick.
• Low resolution ( mostly used
for DNA separation).
( DNA single base pair = 650 Da)
POLYACRYLAMIDE
• Runs vertically.
• Sets by a chemical reaction
once crosslinking occurs.
• Porosity is dictated by acryl
amide concentration
• DNA fragments of 5-500 bp
in size & separate proteins of
5-200 kDa.
• Gel is thin.
• High resolution ( Protein
separation)
( 1 Amino acid= 100 Da)
4

5. GEL CONDITIONS
DENATURING GEL
• Biomolecule's natural
structure is disturbed
Nucleic acid by Urea
RNA by DMSO & glyoxal
Proteins by SDS
• Reducing conditions by beta-
mercaptoethanol or
dithiothreitol in SDS-PAGE
• Biomolecules are separated
on the basis of charge to
mass ratio.
NATIVE GEL
• Biomolecule's natural
structure is maintained
• No denaturing agent is used
• Biomolecules are separated
on the basis of size, charge
and shape.
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6. • allowing for analysis of only
primary structure
When to use?
• Blotting
• DNA & Protein sequencing
• allowing for analysis of all
four levels of the
biomolecular structure
• State of the sample
• Study of enzymes
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7. FACTORS EFFECTING GEL
ELECTROPHORESIS
Electric
Field
Sample Buffer
Supporting
Medium
Voltage. ( V= IR)
Current
Charge, size and shape
Rate of migration α
1/ size and shape
pH, migration of
compounds
Inert, Viscosity
and Pore size.
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8. GEL ELECTROPHORESIS APPARATUS
GEL TANK
POWERSUPPLY COMBSGEL CASTING TRAY
RUBBER END CAP
NEGATIVE
ELECTRODE POSITIVE
ELECTRODE
GLASS SLIDES
TANK LID
8

9. AGAROSE GEL ELECTROPHORESIS FOR THE
SEPRATION OF DNA FRAGMENTS
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10. • Composition: Tris base +
acetic acid+ EDTA
• Large pieces of DNA
• Low voltages, i.e. <150V
• Lower buffering capacity
• Low cost
• Composition: Tris base+ boric
acid + EDTA.
• Small DNA fragments (0.1to
3kb).
• High voltages, i.e. 2000V.
• Higher buffering capacity
• High cost
TAE BUFFER TBE BUFFER
RUNNING BUFFER
REAGENTS & CHEMICALS
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11. GEL TRACKING DYES
Bromophenol blue and Xylene Cyanol.
Density ( Sucrose or glycerol)
DNA STAINING DYE
Ethidium Bromide
EtBr
Orange G
Bromophenol
Blue
Cresol Red
Xylene
Cyanol
Agarose ( 0.5- 2%)
Mix agarose powder with buffer
(TAE/ TBE) in a flask and heat it. Cool
down at 50 °C. Add EtBr to a final
concentration of sol
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12. A- Insert the tray into the groves
of casting dam
B-Insert the comb into the
required position within the tray.
Prepare the agarose gel and pour
into the tray
C-Once the gel becomes opaque
carefully removes the casting
dams before placing the gel
within the tank. Gently Remove
the combs.
D-Pour buffer ( to cover the gel
and fill the wells
E-Comb may be inserted upside
down and reinserted into the
tray to provide convenient
loading support.
Red tape on underside of the
tray or on the gel tank platform
helps well detection.
F-Remove the comb and place
the lid.
Pictures credit- Cleaver Scientific
Limited.
A
D C
B
F E
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13. DNA
MARKER
Sample DNA
BANDS
Smaller
fragment
Larger fragment
Lanes
DNA VISUALIZATION ON TRANSILLUMINATOR
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14. PROTEIN SEPARATION BY SDS-PAGE
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15. SDS is an anionic detergent that denatures
secondary and non–disulphide linked tertiary
structures, and additionally applies a negative
charge to each protein in proportion to its mass.
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16. REAGENTS & CHEMICALS
• Acrylamide
• Methylene bis acrylamide
• Tris HCL
• Sodium dodecyl sulfate ( SDS)
• Ammonium peroxide sulfate ( APS)
• TEMED ( tetramethylethylenediamine)
• Glycine
• 2-mercaptoethanol
• Bromophenol Blue
• Coomassie Brilliant Blue
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17. PREPARATION OF SOLUTIONS
• Acrylamide-bis- acrylamide Sol ( 30:0.8)
• Resolving Gel Buffer ( Tris HCL , pH :8.8)
• Stacking Gel Buffer ( Tris HCL , pH :6.8)
• SDS Solution ( 1 %)
• APS Solution ( 1.5 %)
• Reservoir Buffer ( Tris + Glycine+ SDS , pH :8.3)
• Sample Diluting Buffer ( Tris HCl+ SDS+ 2- mercaptoethanol+
glycerol or sucrose+ Bromophenol blue crystals)
• Staining Solution ( Coomassie blue +Acetic acid + Methanol)
• Destaining Solution ( Acetic acid + Methanol)
• Dissolve protein in sample diluting buffer and heat it in boiling
water bath for 3-4 mins.
All the solutions are prepared using distilled water.
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18. STACKING & RESOLVING OF PROTEINS
RESOLVING GEL
STACKING GEL
1
2
3
Glycine ( N/-)
Proteins ( -)
Cl -
Cl -
Glycine ( N/-)
Proteins ( -)
pH: 6.8
pH: 8.8
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19. PROCEDURE
A- Gather all the
materials.
B- Assemble the gel
casting mold
C- Pour acrylamide
solution. Overlay
with water Allow it
to polymerize for 20-
30 minutes .Remove
the overlaid water.
D- Pour acrylamide-
bis-acrylamide sol
for stacking gel and
insert a comb. Allow
it to polymerize
D
A B
C
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20. E- Remove the comb ,
spacer and the binder
clip. Place the gel in
vertical assembly and
fill it with running
buffer
F- Remove bubbles by
using syringe
G- Load sample into the
wells
H- Turn on the power
supply and run the gel.
H
E
G
F
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21. VISUALIZATION OF PROTEIN BANDS
STAINING SOLUTIONDESTAINING SOLUTION
PROTEIN BANDS
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22. APPLICATIONS OF GEL ELECTROPHORESIS
PCR
DNA
FINGERPRINTING
DNA SEQUENCING
PROTEOMICS BLOTTING
EVOLUTIONARY
RELATIONSHIP
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23. FOR ANY QUERY YOU CAN LEAVE YOUR QUESTION IN THE
COMMENT SECTION
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24. REFERENCES
• Smisek, D. L., Hoagland, D. A. (1989). "Agarose gel electrophoresis of high
molecular weight, synthetic polyelectrolytes". Macromolecules. 22 (5),
2270–2277.
Reece, J. B., Taylor, M. R., Simon, E. J., and Dickey, J. L. (2012). Gel
electrophoresis sorts DNA molecules by size. In Campbell biology: Concepts
& connections (7th ed., p. 243).
• Sambrook J, Russel DW (2001). Molecular Cloning: A Laboratory Manual 3rd
Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.
• Kratz, R. F. and Siegfried, D. R. (2010). Using gel electrophoresis to separate
molecules. In Biology for dummies (2nd ed., pp. 132-133). Hoboken, NJ:
John Wiley & Sons.
Oswald, N. (2008, August 6).
• Lopachin. R. (2004). "The changing view of acrylamide neurotoxicity".
Neurotoxicology . 25 (4): 617–30.
• The principle and method of polyacrylamide gel electrophoresis (SDS-
PAGE). Retrieved from
https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html
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25. • Fitzpatrick, Richard. (2007) “Electric Fields.” The University of Texas at
Austin.
• How To Cast And Run An Agarose Gel in The Multi Sub Mini Electrophoresis
System Retrieved from https://youtu.be/zXgM10ghY_w
• How to stain an SDS- PAGE Gel. Retrieved from https://youtu.be/b-
1dXzU4iOw.
• http://www.cpet.ufl.edu/wp-content/uploads/2013/10/Intro-Gel-
Electrophoresis-manual.pdf
• https://webfiles.uci.edu/treseder/public/Protocols/Agarose%20Gel%20Elec
trophoresis.pdf
• https://openwetware.org/wiki/Agarose_gel_loading_buffer
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26. PICTURES CREDIT
• https://www.enasco.com/p/Edvotek-M12-Dual-Gel-Electrophoresis-
Apparatus%2BSB45606
• https://www.fishersci.co.uk/shop/products/tv100-standard-cooled-twin-plate-
mini-gel-electrophoresis-unit/15805551
• https://www.mikeblaber.org/oldwine/BCH4053l/Lecture05/Lecture05.htm
• https://www.google.com/url?sa=i&url=https%3A%2F%2Fgeneticeducation.co.in
%2Frole-of-etbr-in-agarose-gel-electrophoresis-
karyotyping%2F&psig=AOvVaw0LTwrmtAPixWpKyLQFU-
AG&ust=1596730659749000&source=images&cd=vfe&ved=
• https://openwetware.org/wiki/Xylene_cyanol
• https://molbio.mgh.harvard.edu/szostakweb/protocols/protein_page/index.ht
ml
• https://en.wikipedia.org/wiki/Polyacrylamide
• https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html
• http://www.protocol-online.org/biology-forums-2/posts/17700.html
• https://askabiologist.asu.edu/agarose-gel-electrophoresis
• https://videorista.com/index.php?route=product/product&product_id=62
• https://en.wikipedia.org/wiki/Protein
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