Gel Electrophoresis: Introduction and Techniques Martin Cole (isoelectric focusing), Mcolisi Dlamini, Faraz Khan April 18. 2012 Physics 200: Molecular

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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
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  • 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
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  • 1920s Erich Huckel and M. Smoluchowski are among the pioneers of electrophoresis. Huckel developed the Huckel equation D. C Henry provided a theory spherical polyions. 1930s A. Tiselius: Nobel Prize for Chemistry in 1948 Introduced idea of moving boundaries 1960s 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: Overview 1
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  • 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. Kargers 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
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  • Parts of the System Gel Support Medium Agarose Polyacrylamide (PA) Native Gels Use PA or Starch No Denaturant Buffer DC Power Supply
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  • Basics www.davidson.edu/academic/biology/courses/molbio/sdspage/sdspage.html
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  • Molecule in an Electric Field http://web.ncf.ca/ch865/englishdescr/EFld2Plates.html E Q+ QE f*u
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  • Deriving u INDEX Q = charge E = Electric field m = mass f = friction coefficient u = velocity
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  • Electrophoretic Mobility,
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  • Units of http://eculator.com/formula/calculator.do?equation=Capacitance-of-parallel-plate-capacitor&id=41
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  • Does not correspond to Reality, Not done! https://www.mecheng.osu.edu/cmnf/what-micro-and-nano-fluidics
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  • Huckel Equation
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  • Electrophoretic Experiments MethodNotes Moving Boundary Electrophoresis or Free Electrophoresis -Gives mobility -Basis: particles transport properties Thin layer Zone or Zonal gel Electrophoresis -Uses a matrix as a sieve to separate molecules -Basis: size -Gel: provides stability against convection Electric birefringence-Not in syllabus
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  • Free Electrophoresis Electrophoretic 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/
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  • Forces on the Particle
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  • 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
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  • Electrophoretic Mobility Smoluchowski Determined another way to view electrophoretic mobility 2 Only for Thin double layer http://en.wikipedia.org/wiki/Marian_Smoluchowski
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  • (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
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  • Hckel Correction Smoluchowski did not correct for Debye length Length over which charges are screened 3 Denoted by
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  • http://www.silver-colloids.com/Tutorials/Intro/pcs21.html
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  • 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
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  • Steady State Electrophoresis
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  • Support Medium Electrophoresis Agarose Starch SDS-PAGE Native Set up http://www.aesociety.org/areas/preparative_gel.php
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  • 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
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  • SDS-PAGE 6 SDS Sodium Dodecyl Sulfate Denaturant Movement based only on molecular mass -mercaptoethanol PAGE Polyacrylamide Support http://www.davidson.edu/academic/biology/courses/molbio/sdspage/sdspage.html
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  • SDS-PAGE http://www.youtube.com/watch?v=IWZN_G_pC8U
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  • 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
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  • 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
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  • 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.