Gel Electrophoresis: Introduction and Techniques

1. Gel Electrophoresis: Introduction and Techniques Martin Cole (isoelectric focusing), McolisiDlamini, Faraz Khan April 18. 2012 Physics 200: Molecular Biophysics http://vadlo.com/cartoons.php?id=445

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. 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 • 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 E f*u Q+ QE http://web.ncf.ca/ch865/englishdescr/EFld2Plates.html

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

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

14. 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

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 • β-mercaptoethanol • 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 • 1Serdyuk, 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.