Gel Electrophoresis Introduction and Techniques http vadlo comcartoons

Gel Electrophoresis: Introduction and Techniques http: //vadlo. com/cartoons. php? id=445 Martin Cole (isoelectric focusing), Mcolisi Dlamini, Faraz Khan April 18. 2012

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 2 D 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. html

Molecule in an Electric Field E f*u http: //web. ncf. ca/ch 865/englishdescr/EFld 2 Plates. html Q+ QE

Deriving u � INDEX Q = charge E = Electric field m = mass f = friction coefficient u = velocity

Electrophoretic Mobility, μ �

Units of μ http: //eculator. com/formula/calculator. do? equation=Capacitance-of-parallel-plate-capacitor&id=41 �

Does not correspond to Reality, Not done! � https: //www. mecheng. osu. edu/cmnf/what-micro-and-nano-fluidics

Huckel Equation �

Electrophoretic Experiments Method Notes 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

Free Electrophoresis �Electrophoretic separation without gel support ◦ Capillary electrophoresis ◦ Free Flow Electrophoresis http: //www. utwente. nl/ewi/bios/research/micronanofluidics/old micro-nanofluidicsprojects/Microfluidic/ http: //www. youtube. com/watch? feature=player_detailpage&v=ln. Ac. Vi. Ysz 4 g#t=

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 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/pcs 21. html

Steady State Electrophoresis �Ions trapped and sealed with semipermeable membrane �Electric Field ◦ Flux of ions �Steady State ◦ Fluxes of ion and electric field equal http: //www. spinanalytical. com/mce-productstheory. 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 nondenatured proteins http: //delliss. people. cofc. edu/virtuallabbook/Loading. Gel. 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. html

SDS-PAGE http: //www. youtube. com/watch? v=IWZN_G_p. C 8 U

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/lecture 6/index. ht m

Viewing Conditions �Staining depends on type of molecule �View Under UV �DNA ◦ Ethidium Bromide ◦ Gel. Red �Protein ◦ Coomassie Brilliant Blue ◦ Horse Radish Peroxidase http: //www. biotium. com/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/b 241/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.