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Gel Electrophoresis: Introduction and Techniques
Martin Cole (isoelectric focusing), Mcolisi Dlamini, Faraz Khan
April 18. 2012
Physics 200: Molecular Biophysics
http://vadlo.com/cartoons.php?id=445
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
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
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 SystemGel 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
http://web.ncf.ca/ch865/englishdescr/EFld2Plates.html
E
Q+ QEf*u
Deriving u
a=0, then
INDEX
Q = charge
E = Electric field
m = mass
f = friction coefficient
u = velocity
Electrophoretic Mobility, μDefined as the ratio of the
particles velocity to the strength of the driving field.
Therefore,
- Now the velocity depends on the particle properties.
Units of μ
[𝝁 ]=𝒄𝒎𝟐
𝑽𝒔
So,
Therefore,
http://eculator.com/formula/calculator.do?equation=Capacitance-of-parallel-plate-capacitor&id=41
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
Huckel EquationUsed to model electrostatic mobility.Assume that the particle is a sphere, then Stokes equation applies.
Electrophoretic Experiments
Method Notes
Moving Boundary ElectrophoresisorFree Electrophoresis
- Gives mobility- Basis: particles transport
properties
Thin layer Zoneor 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 ElectrophoresisElectrophoretic
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/
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 MobilitySmoluchowski
◦ 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 CorrectionSmoluchowski
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 ElectrophoresisIons 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
Steady State Electrophoresis
Support Medium ElectrophoresisAgarose StarchSDS-PAGENative Set up
http://www.aesociety.org/areas/preparative_gel.php
Agarose and Starch GelsAgarose
◦ 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 ConditionsUse PA supportNo 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 ConditionsStaining depends
on type of molecule
View Under UVDNA
◦ 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 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.