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: Overview

1    ◦ ◦

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

f*u

E Q+ QE http://web.ncf.ca/ch865/englishdescr/EFld2Plates.html

Deriving u

𝐹 𝑟𝑖𝑔ℎ𝑡 𝐹 𝑙𝑒𝑓𝑡 = 𝑄𝐸 = 𝑓𝑢 a=0, then 𝐹 𝑛𝑒𝑡 = 𝑚𝑎 𝑄𝐸 = 𝑓𝑢 𝑢 = 𝑄𝐸 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 http://eculator.com/formula/calculator.do?equation=Capacitance-of-parallel-plate-capacitor&id=41 𝝁 = 𝒄𝒎 𝟐 𝑽𝒔 𝑉 = 𝐸𝑑 So, 𝑉 𝐸 = Therefore, 𝑑 𝑉 𝐸 = 𝑐𝑚

Does not correspond to Reality, Not done!

1.

2.

Net charge – due to counterions. Net charge is used instead.

𝑄 𝑒𝑓𝑓 𝜇 = 𝑓 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

Moving Boundary Electrophoresis or Free Electrophoresis Thin layer Zone or Zonal gel Electrophoresis Electric birefringence

Notes

- Gives mobility - Basis: particles transport properties - Uses a matrix as a sieve to separate molecules - Basis: size - Gel: provides stability against convection - 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

   F HD ◦ Hydrodynamic Friction F CF ◦ Counter ion Flow ◦ Particle Travels Upstream F FA ◦ Field Asymmetry Effect http://www.websters-online-dictionary.org/definitions/Electrophoresis

Electrophoretic Mobility

 Smoluchowski ◦ Determined another way to view electrophoretic mobility 2 ◦ 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 screened 3 Denoted by ◦ κ

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

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

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 separations 4 ◦ Can easily be stored for tagging 5 Starch ◦ Also used to separate non-denatured proteins http://delliss.people.cofc.edu/virtuallabbook/LoadingGel/LoadingGel.html

SDS-PAGE

6   ◦ ◦ 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 Charge 7 ◦ 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

       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.

7 Arakawa, 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.