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.