Gel Electrophoresis:
Introduction and Techniques
http://vadlo.com/cartoons.php?id=445
Martin Cole (isoelectric focusing), Mcolisi Dlamini, Faraz Khan
April 18. 2012
Physics 200: Molecular Biophysics
What does it do?

Separation of
◦ Proteins
 Western Blots
 SDS-PAGE
◦ Nucleic Acids
 Northern Blots
 Southern Blots

Based on
◦ Charge and/or
◦ Size

What else?
◦ Torture Undergrads
History: Overview1
1920’s
◦ Erich Huckel and M. Smoluchowski are among the
pioneers of electrophoresis.
◦ Huckel developed the Huckel equation
◦ D. C Henry – provided a theory spherical
polyions.
 1930’s
◦ A. Tiselius: Nobel Prize for Chemistry in 1948
 Introduced idea of moving boundaries
 1960’s
◦ A. L. Shapiro, E. Vinuela and J. V. Maizel: developed
relationship between electrophoretic migration of
proteins and their molecular weight.

Erich Huckel
Arne Tiselius
History: Overview

1975
◦ Farrell and J. Klose: developed 2D electrophoresis

1981
◦ J. W. Jorgensen and K. D. Lukas: performed electrophoretic amino
acid separation at high efficiency

1990
◦ B. L. Karger’s group: discovered a matrix that could be used to
separate DNA at high resolution

All these improvements led to the use of electrophoresis in
mapping the human genome.

2000 to now
◦ widely used high-resolution techniques for analytical and
preparative separations
Parts of the System

Gel Support Medium
◦ Agarose
◦ Polyacrylamide (PA)
◦ Native Gels
 Use PA or Starch
 No Denaturant
Buffer
 DC Power Supply

Basics
www.davidson.edu/academic/biology/courses/molbio/sdspage/sdspage.html
Molecule in an Electric Field
E
f*u
http://web.ncf.ca/ch865/englishdescr/EFld2Plates.html
Q+
QE
Deriving u
𝐹𝑟𝑖𝑔ℎ𝑡 = 𝑄𝐸
𝐹𝑙𝑒𝑓𝑡 = 𝑓𝑢
𝐹𝑛𝑒𝑡 = 𝑚𝑎
a=0, then
𝑄𝐸 = 𝑓𝑢
𝑄𝐸
𝑢=
𝑓
INDEX
Q = charge
E = Electric field
m = mass
f = friction
coefficient
u = velocity
Electrophoretic Mobility, μ
Defined as the ratio of the particles
velocity to the strength of the driving
field.
𝑢
𝜇=
𝐸
Therefore,
𝑄
𝜇 = ⇒ 𝑄 = 𝜇𝑓
𝑓
- Now the velocity depends on the particle
properties.

Units of μ
𝑉 = 𝐸𝑑
So,
𝑉
𝐸=
𝑑
Therefore,
𝑉
𝐸 =
𝑐𝑚
http://eculator.com/formula/calculator.do?equation=Capacitance-of-parallel-plate-capacitor&id=41
𝒄𝒎𝟐
𝝁 =
𝑽𝒔
Does not correspond to Reality, Not done!
1.
Net charge – due to counterions. Net charge is
used instead.
𝑄𝑒𝑓𝑓
𝜇=
𝑓
2.
Convection effects – corrected by using gels
https://www.mecheng.osu.edu/cmnf/what-micro-and-nano-fluidics
Huckel Equation
Used to model electrostatic mobility.
𝑍𝑒
𝜇=
𝑓
Assume that the particle is a sphere, then Stokes
equation applies.
𝑍𝑒
𝜇=
6𝜋𝜂𝑅
Electrophoretic Experiments
Method
Notes
Moving Boundary Electrophoresis
or
Free Electrophoresis
- Gives mobility
- Basis: particles transport properties
Thin layer Zone
or
Zonal gel Electrophoresis
- Uses a matrix as a sieve to separate
molecules
- Basis: size
- Gel: provides stability against
convection
Electric birefringence
- Not in syllabus
Free Electrophoresis

Electrophoretic
separation without
gel support
◦ Capillary
electrophoresis
◦ Free Flow
Electrophoresis
http://www.utwente.nl/ewi/bios/research/micronanofluidics/oldmicro
-nanofluidicsprojects/Microfluidic/
http://www.youtube.com/watch?feature=player_detailpage&v=lnAcViYsz4g#t=161s
Forces on the Particle
Retardation Forces

FHD
◦ Hydrodynamic
Friction

FCF
◦ Counter ion Flow
◦ Particle Travels
Upstream

FFA
◦ Field Asymmetry Effect
http://www.websters-online-dictionary.org/definitions/Electrophoresis
Electrophoretic Mobility

Smoluchowski
◦ Determined another
way to view
electrophoretic
mobility2
◦ Only for Thin double
layer
http://en.wikipedia.org/wiki/Marian_Smoluchowski
ξ (Zeta Potential)
Electric potential in
the double layer
 Potential difference
between dispersion
medium and cage
around particle
 Important in stability
of particles

http://en.wikipedia.org/wiki/Zeta_potential
Hückel Correction

Smoluchowski did
not correct for
Debye length
◦ Length over which
charges are screened3

Denoted by
◦ κ
http://www.silver-colloids.com/Tutorials/Intro/pcs21.html
Steady State Electrophoresis
Ions trapped and
sealed with semipermeable
membrane
 Electric Field

◦ Flux of ions

Steady State
◦ Fluxes of ion and
electric field equal
http://www.spinanalytical.com/mce-products-theory.php
Steady State Electrophoresis
Support Medium Electrophoresis
Agarose
 Starch
 SDS-PAGE
 Native Set up

http://www.aesociety.org/areas/preparative_gel.php
Agarose and Starch Gels

Agarose
◦ Used in DNA
separation methods
◦ Can be sued in Large
protein separations4
◦ Can easily be stored
for tagging5

Starch
◦ Also used to separate
non-denatured
proteins
http://delliss.people.cofc.edu/virtuallabbook/LoadingGel/LoadingGel.html
SDS-PAGE6

SDS
◦ Sodium Dodecyl
Sulfate
◦ Denaturant
◦ Movement based only
on molecular mass
◦ β-mercaptoethanol

PAGE
◦ Polyacrylamide
Support
http://www.davidson.edu/academic/biology/courses/molbio/sdspage/sdspage.html
SDS-PAGE
http://www.youtube.com/watch?v=IWZN_G_pC8U
Native Gel Conditions
Use PA support
 No Denaturant

◦ Protein stays in
original conformation
◦ Protect from
Oxidation

Movement depends
on:
◦ Intrinsic Charge7
◦ Hydrodynamic Size
http://ccnmtl.columbia.edu/projects/biology/lecture6/index.htm
Viewing Conditions
Staining depends on
type of molecule
 View Under UV
 DNA

◦ Ethidium Bromide
◦ GelRed

Protein
◦ Coomassie Brilliant
Blue
◦ Horse Radish
Peroxidase
http://www.biotium.com/product/product_types/search/price_and_info.asp?item=41003
References
Serdyuk, I., Zaccai, N., & Zaccai, J. (2007). Methods in Molecular Biophysics: Structure, Dynamics,
Function. Cambridge: Cambridge University Press.

1

2 von

3 Huckel, E. (1924). Physik. Z.

4 Smisek, D., &

5 Massachusets

6 Voet, D., Voet, J., &
Smoluchowski, M. (1903). Bulletin International de l'Academi des Sciences de Cracovie , 184.
(25), 204.
Hoagland, D. (1989). Agarose Gel Electrophoresis of high molecular weight,
synthetic polyelectrolytes. Macromolecules , 22 (5.), 2270-2277.
Institute of Technology. (n.d.). Essential Techniques of Molecular Genetics. Retrieved
2012, from MIT Biology Hypertextbook: http://www.ucl.ac.uk/~ucbhjow/b241/techniques.html
Pratt, C. (2008). Fundamentals of Biochemistry: Life at the Molecular Level.
Hoboken: Wiley.

7Arakawa, T., Philo, J., Ejima, D., Tsumoto, K., & Arisaka, F. (2006). Aggregation
analysis of
therapeutic proteins, part 1: General aspects and techniques for assessment. Bioprocess
International , 4 (10), 42-49.