Gel Electrophoresis

Introduction
• from electro- + Greek phorēsis ‘being carried’.
• The first electrophoretic separation technique by Tiselius,
1937
• Moving boundary electrophoresis.
• Received Nobel Prize 1948 Fig 1: Arne Wilhelm
Kaurin Tiselius

• The number of techniques increased dramaticaly

• High separation efficiency with relatively simple equipment
• Integral part of modern biology

Tiselius, A. (1937). Transactions of the Faraday Society. 33: 524–1933.

 Gel

𝐹1 __
𝐹2
Principle E

• 𝐹1 = 𝑞𝐸 (Force from electric field)

𝑉
• 𝐹1 = 𝑞
𝑑
• 𝐹2 = 6𝜋𝑟𝜂𝑣 (Viscous force, Stoke’s equation)
𝑞𝑉 𝑞𝐸
•𝑣= =
6𝜋𝑑𝑟𝜂 6𝜋𝑟𝜂
Gel Electrophoresis
• Electrophoresis in which a gel is used as a restrictive media.
• Main components
• Gel
• Buffers
• Sample
• Power Supply
• Tank
The gel
• Important features of a gel
• Adjustable and regular pore size
• Chemicaly inert
• No electroendosmosis
• Electroendosmosis
• Caused by charged groups part of the gel
• Eg COO- fixed in the matrix
• Cause counter flow of H3O+ ions to cathod.
• Cause blurred Zones
Types of gel
Starch gel- Smithies(1955)
• From potato starch.
• Pore size adjusted by starch concentration
• Low reproducibility
Agarose Gel- Bockemuller W, Oerter A(1956)
• Polysaccharide btained from seaweed
• Large pore size for molecules with diameter over 10nm
• Pore size depends on Agarose concentration
• 0.16% -> 500nm and 1 % gives 150nm

Smithies O. Biochem J. 61 (1955) 629–641.

BOCKEMULLER, W., & OERTER, A. (1956)., dem Georg-Speyer-Haus und dem Ferdinand-Blum-Institut zu Frankfurt a.M, 52, 61-79.
https://www.permaculture.co.uk/news/1503121560/greek-potato-revolution-spreads-economic-hardship-relocalises-food-supply
Types of Gel
Agarose Gel
• A disaccharide
• Dissolves in boiling water
and forms a polymer in
cooling
• 1->3 and 1->4 glyosidic
bond
• Forms random coils on
cooling which laterally join
to form thick filaments via
H-H bonds
Wujie Zhang (2015). Encapsulation of Transgenic Cells for Gene Therapy, Gene
Therapy - Principles and Challenges, Dr. Doaa Hashad (Ed.), InTech
Types of Gel
Types of gel
Polyacrylamide gel- Raymond and Weintraub
(1959)
• Chemicaly inert and mechanically stable
• Co-polymerisation of acrylamide monomers with
Crosslinking agent N,N’-methylenebisacrylamide
• Free-radical polymerisation reaction
• Persulphate salt provide starting free radical
• The pore size can controlled by the total
acrylamide concentration T and the degree of
cross-linking C (Hjertn, 1962):

Raymond S. Weintraub L. Science. 130 (1959) 711–711.

Hjertn S. Arch Biochem Biophys Suppl 1 (1962) 147.
Polyacrylamide gel
Agarose gel electrophoresis
For Protein
• Proteins need competitively smaller pore size
than DNA
• Agarose gel >1% are cloudy
• Thus only high MW proteins and protein
aggregates are seperated using agarose gel
Agarose gel electrophoresis
For Nucleic acid
• Standard method for separation, restriction fragment analysis, and
purification
• Horizontal gel lies directly submerged in buffer.
Agarose gel electrophoresis
•Buffer
•Tris Borate EDTA and Tris-Acetate-EDTA Buffer (TBE &
TAE), pH 8.3
•Tris with borate and acetate maintain pH, EDTA chelate
cations
•Longer fragments run faster on TAE and Gel elution yield
is more.
•But TBE gives more resolution

http://www.aniara.com/pdf/Tris-Ace-EDTA-Buf.pdf
Agarose gel electrophoresis
•Sample Preparetion
•Sample containing DNA or RNA is mixed with loading dye.
•Loading dye(6X) usually contain
• 30% (v/v) glycerol
• 0.25% (w/v) bromophenol blue
• 0.25% (w/v) xylene cyanol FF
• In water
•Function of loading dye- Prevent diffusion & Dyes give an idea of
extend of gel run

doi:10.1101/pdb.rec11045Cold Spring Harb Protoc2007.

Agarose gel electrophoresis
•Imaging
•Gel stained with intercalating Fluorescent dyes like Ethidium bromide OR
SYBR Green
•Bands are visualised under uv
•Sensitivity 100 pg to1 ng / band.(lower for RNA)

http://www.rosesci.com/Products/products.php?title=Electrophoresis%20Systems,%20the%20Major
%20Science%20SafeBlue,%20with%20Blue%20Light%20Leds%20EE
https://pubchem.ncbi.nlm.nih.gov
Pulsed field Gel Electrophoresis (PFG)
• Schwartz and Cantor (1984)
• For High molecular weight DNA molecules over 20 kb
• Eg Chromosome
• Normally DNA <20 Kb migrate with same mobility
• As they align themselves lengthwise
• In PFG
• There is change in direction of Electric field
• The helical structure get stretched and compressed
• Reorientation takes time, more for larger molecules
• Separation is obtained up to 10MBp

Schwartz DC, Cantor CR. Cell. 37 (1984) 67–75.

Pulsed field Gel Electrophoresis (PFG)
•Used in Bacterial Taxonomy
•Method
•1.0 to 1.5% agarose gels are used
•The electric fields should have an angle of at
least 110⁰ relative to the sample.
•obtained by using point electrodes mounted
on orthogonal rails or in hexagonal
configuration.
•The pulse time is of 1 s to 90 min
•Larger molecules separate with longer pulse
time
Polyacrylamide gel electrophoresis
For Nucleotides
• Sharper bands and higher resolution ~ 15pg/
Band by Silver staining
• High separation up to 1 bp
•Used mainly for DNA Sequencing
•Dissadvantage
•Sequence influence mobility of fragments, AT rich
migrate slower
Polyacrylamide gel electrophoresis
For Protein
•Thin flat gel used now
•Protein aggregate and and precipitate of proteins during the entry from
liquid sample into the gel matrix and results in blurry bands
•Ornstein (1964) and Davis (1964) suggested Discontinuous gel prevents this
•Gel has two areas
•Resolving gel
•Stacking gel

Ornstein L. Ann NY Acad Sci.121 (1964) 321–349.

Davis BJ. Ann NY Acad Sci.121 (1964) 404–427.
Polyacrylamide gel electrophoresis
•Stacking gel
•large pores
•0.125 mol/L Tris-HCL pH 6.8
•Electrode buffer has glycine- almost no net charge at 6.8->
Trailing anion
•𝐶𝑙 − has high charge ->Leading cation
• Protein form stack by Isotachophoresis
•Separation not dependent on mass
Polyacrylamide gel electrophoresis
•Resolving gel
•Smaller pore
•0.375 mol/L Tris-HCL buffer pH 8.8
•Protein stack frictional resistance increase
•Become concentrated
•𝐶𝑙 − And 𝐺𝑙𝑦𝑐𝑖𝑛𝑒 − front move towards cathode
1. No more trailing anion
•Thus destak by Zone electrophoresis
2. Mobility depends on charge and mass
3. pH rise to 9.5 -> Seperation faster
Polyacrylamide gel electrophoresis
Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
•Shapiro et al. (1967)
•SDS -> anionic detergent SDS
•Makes charge per mass constant
•Molecule denatured by SDS and 2-mercaptoethanol

Shapiro AL, Viuela E, Maizel JV. Biochem Biophys Res Commun. 28 (1967) 815–822.
SDS-PAGE
Chemical structure SDS

•Advantages
•SDS solubilizes almost all proteins
•Since SDS-protein complexes are highly charged
•All fragments migrate in one direction.
•Separation only based on MW
•Usually done using protocol developed by Lammli in 1970

Lammli UK. Nature 227 (1970) 680–685.

https://pubchem.ncbi.nlm.nih.gov
SDS PAGE
Polyacrylamide gel electrophoresis

•Staining
•Coomassie
•Few steps
•Staining soln with methanol acetic acid and
Coomassie G-250
•Destaining soln with metnanol and acetic acid
•Silver Staining(Merril CR, et al., 1981)
•𝐴𝑔𝑁𝑂3 gets easily reduced to metallic Ag
•𝐴𝑔2+ Binds to proteins
•Reducing agent is used after wash to stain
protein
•Westen blot
Merril CR, et al. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in
cerebrospinal fluid proteins. Science. 1981;211:1437–1438.
Adam V, et al.Sensors (Basel). 2008 Jan; 8(1): 464–487
Isoelectric Focusing (IEF)
• Electrophoresis takes place in a pH
gradient made using Ampholytes and
IPG
• Molecules move toward anode or
cathode until they reach aa position
where their net charge is 0 (isoelectric
point)
• Can only be used to separate
amphoteric substances
• Like Peptide and Proteins
IEF-2D PAGE

• 2 dimensions of separation
1. Zone electrophesis
2. Isoelectric focusing
•Or Vive versa
•Gives an overall picture of the protein composition
•Enable location of individual proteins
High resolution 2-D electrophoresis
-> O’Farrell (1975)
•The sample is denatured
with a lysis buffer
•First dimension is
isoelectric focusing in
presence of 8 or 9 molar
urea and a non-ionic
detergent
•SDS electrophoresis is
run as the second
dimension.

O’Farrell PH. J Biol Chem. 250 (1975) 4007–4021.

High resolution 2-D electrophoresis
•Very useful method
•Protein spots can be analysed by Mass Spectroscopy
•Short Sequence can be found
•Can identify locus in the genome
Key Reference
1. Westermeier, R., 2005. Electrophoresis in Practice, Fourth, revised
and enlarged edition. Wiley-VCH, Weinheim.
2. Cooper, T., G., 1977. The Tools of Biochemistry. John Wiley &
Sons, Hoboken.
3. Berkelman, T., Stenstedt, T., 1998. 2-D Electrophoresis using
immobilized pH gradients principles & methods
Thank You
 See Hidden slides for more info
on Stacking and Denaturing Page

Coomassie Staining mechanism

doi:10.1038/nprot.2009.214