1. Gel Electrophoresis: Introduction and Techniques
Martin Cole (isoelectric focusing), Mcolisi Dlamini, Faraz Khan
April
Physics 200: Molecular Biophysics

2. What does it do? Separation of Based on What else? Proteins
Western Blots
SDS-PAGE
Nucleic Acids
Northern Blots
Southern Blots
Based on
Charge and/or
Size
What else?
Torture Undergrads

3. 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

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 Buffer DC Power Supply Agarose
Polyacrylamide (PA)
Native Gels
Use PA or Starch
No Denaturant
Buffer
DC Power Supply

6. Basics

7. Molecule in an Electric Field
f*u Q+ QE

8. Deriving u 𝐹 𝑟𝑖𝑔ℎ𝑡 =𝑄𝐸 𝐹 𝑙𝑒𝑓𝑡 =𝑓𝑢 𝐹 𝑛𝑒𝑡 =𝑚𝑎 a=0, then 𝑄𝐸=𝑓𝑢 𝑢= 𝑄𝐸 𝑓
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.
𝜇= 𝑢 𝐸
Therefore,
𝜇= 𝑄 𝑓 ⇒𝑄= 𝜇𝑓
- Now the velocity depends on the particle properties.

10. Units of μ 𝑉=𝐸𝑑 So, 𝐸= 𝑉 𝑑 Therefore, 𝐸 = 𝑉 𝑐𝑚 𝝁 = 𝒄 𝒎 𝟐 𝑽𝒔
𝝁 = 𝒄 𝒎 𝟐 𝑽𝒔

11. Does not correspond to Reality, Not done!
Net charge – due to counterions. Net charge is used instead.
𝜇= 𝑄 𝑒𝑓𝑓 𝑓
Convection effects – corrected by using gels

12. Huckel Equation
Used to model electrostatic mobility. 𝜇= 𝑍𝑒 𝑓 Assume that the particle is a sphere, then Stokes equation applies. 𝜇= 𝑍𝑒 6𝜋𝜂𝑅

13. Electrophoretic Experiments
Method
Notes
Moving Boundary Electrophoresis or
Free Electrophoresis
Gives mobility
Basis: particles transport properties
Thin layer Zone
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

15. Forces on the Particle

16. Retardation Forces FHD FCF FFA Hydrodynamic Friction Counter ion Flow
Particle Travels Upstream
FFA
Field Asymmetry Effect

17. Electrophoretic Mobility
Smoluchowski
Determined another way to view electrophoretic mobility2
Only for Thin double layer

18. ξ (Zeta Potential) Electric potential in the double layer
Potential difference between dispersion medium and cage around particle
Important in stability of particles

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

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

22. Steady State Electrophoresis

23. Support Medium Electrophoresis
Agarose
Starch
SDS-PAGE
Native Set up

24. Agarose and Starch Gels
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

25. SDS-PAGE6 SDS PAGE Sodium Dodecyl Sulfate Denaturant
Movement based only on molecular mass
β-mercaptoethanol
PAGE
Polyacrylamide Support

26. SDS-PAGE

27. Native Gel Conditions Use PA support No Denaturant
Protein stays in original conformation
Protect from Oxidation
Movement depends on:
Intrinsic Charge7
Hydrodynamic Size

28. Viewing Conditions Staining depends on type of molecule View Under UV
DNA
Ethidium Bromide
GelRed
Protein
Coomassie Brilliant Blue
Horse Radish Peroxidase

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.),
5 Massachusets Institute of Technology. (n.d.). Essential Techniques of Molecular Genetics. Retrieved 2012, from MIT Biology Hypertextbook:
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),