Gel electrophoresis

Useful for all science faculty

1. Gel Electrophoresis:
Introduction and Techniques

2. 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

3. 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.
Arne Tiselius
Erich Huckel
History: Overview1

4. 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

5. Parts of the System
Gel Support Medium
◦Agarose
◦Polyacrylamide (PA)
◦Native Gels
◦Use PA or Starch
◦No Denaturant
Buffer
DC Power Supply

6. Basics
www.davidson.edu/academic/biology/courses/molbio/sdspage/sdspage.html

7. Molecule in an Electric Field
http://web.ncf.ca/ch865/englishdescr/EFld2Plates.html
E
Q+ QE
f*u

8. Deriving u
a=0, then
 
INDEX
Q = charge
E = Electric field
m = mass
f = friction
coefficient
u = velocity

9. Electrophoretic Mobility,
μ
Defined as the ratio of the particles
velocity to the strength of the driving
field.
Therefore,
- Now the velocity depends on the particle
properties.
 

10. Units of
μ
 
So,
Therefore,
 
http://eculator.com/formula/calculator.do?equation=Capacitance-of-parallel-plate-capacitor&id=41

11. Does not correspond to Reality, Not
done!
1.Net charge – due to counterions. Net charge is
used instead.
2.Convection effects – corrected by using gels
 
https://www.mecheng.osu.edu/cmnf/what-micro-and-nano-fluidics

12. Huckel Equation
Used to model electrostatic mobility.
Assume that the particle is a sphere, then Stokes
equation applies.
 

13. 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

14. Free Electrophoresis
Electrophoretic
separation without
gel support
◦Capillary
electrophoresis
◦Free Flow
Electrophoresis
http://www.youtube.com/watch?feature=player_detailpage&v=lnAcViYsz4g#t=161s
http://www.utwente.nl/ewi/bios/research/micronanofluidics/oldmicro-
nanofluidicsprojects/Microfluidic/

15. Forces on the Particle

16. Retardation Forces
FHD
◦Hydrodynamic Friction
FCF
◦Counter ion Flow
◦Particle Travels Upstream
FFA
◦Field Asymmetry Effect
http://www.websters-online-dictionary.org/definitions/Electrophoresis

17. Electrophoretic Mobility
Smoluchowski
◦Determined another
way to view
electrophoretic
mobility2
◦Only for Thin double
layer
http://en.wikipedia.org/wiki/Marian_Smoluchowski

18. 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

19. Hückel Correction
Smoluchowski did
not correct for Debye
length
◦Length over which
charges are screened3
Denoted by
◦κ

20. http://www.silver-colloids.com/Tutorials/Intro/pcs21.html

21. Steady State Electrophoresis
Ions trapped and
sealed with semi-
permeable
membrane
Electric Field
◦Flux of ions
Steady State
◦Fluxes of ion and
electric field equal
http://www.spinanalytical.com/mce-products-theory.php

22. Steady State Electrophoresis

23. Support Medium Electrophoresis
Agarose
Starch
SDS-PAGE
Native Set up
http://www.aesociety.org/areas/preparative_gel.php

24. 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

25. SDS-PAGE6
SDS
◦Sodium Dodecyl Sulfate
◦Denaturant
◦Movement based only on
molecular mass
◦-mercaptoethanol
β
PAGE
◦Polyacrylamide Support
http://www.davidson.edu/academic/biology/courses/molbio/sdspage/sdspage.html

26. SDS-PAGE
http://www.youtube.com/watch?v=IWZN_G_pC8U

27. 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

28. 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

29. References
1 Serdyuk, I., Zaccai, N., & Zaccai, J. (2007). Methods in Molecular Biophysics:
Structure, Dynamics, Function. Cambridge: Cambridge University Press.
2 von Smoluchowski, M. (1903). Bulletin International de l'Academi des Sciences de
Cracovie , 184.
3 Huckel, E. (1924). Physik. Z. (25), 204.
4 Smisek, D., & Hoagland, D. (1989). Agarose Gel Electrophoresis of high molecular
weight, synthetic polyelectrolytes. Macromolecules , 22 (5.), 2270-2277.
5 Massachusets 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
6 Voet, D., Voet, J., & 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.