Gel Electrophoresis: Introduction and Techniques - PowerPoint Presentation

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Gel Electrophoresis: Introduction and Techniques

Martin Cole (isoelectric focusing), Mcolisi Dlamini, Faraz KhanApril 18. 2012Physics 200: Molecular Biophysics

http://vadlo.com/cartoons.php?id=445

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What does it do?

Separation ofProteinsWestern BlotsSDS-PAGENucleic AcidsNorthern BlotsSouthern BlotsBased onCharge and/orSizeWhat else?Torture Undergrads

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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’sA. Tiselius: Nobel Prize for Chemistry in 1948

Introduced idea of moving boundaries1960’sA. 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

1975Farrell and J. Klose: developed 2D electrophoresis1981J. W. Jorgensen and K. D. Lukas: performed electrophoretic amino acid separation at high efficiency1990B

. 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

nowwidely

used high-resolution techniques for analytical and preparative separations

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Parts of the System

Gel Support MediumAgarosePolyacrylamide (PA)Native GelsUse PA or StarchNo DenaturantBuffer 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

a=0, then

 

INDEX

Q = charge

E = Electric field

m = mass

f = friction coefficient

u = velocity

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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.

 

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Units of

μ

 

So,

Therefore,

 

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!

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

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Huckel Equation

Used to model electrostatic mobility.

Assume that the particle is a sphere, then Stokes equation applies.

 

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Electrophoretic Experiments

Method NotesMoving Boundary ElectrophoresisorFree Electrophoresis

Gives mobilityBasis: particles transport properties

Thin layer

Zone

or

Zonal gel Electrophoresis

Uses a matrix as a sieve to separate moleculesBasis: sizeGel: provides stability against convectionElectric

birefringenceNot in syllabus

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Free Electrophoresis

Electrophoretic separation without gel supportCapillary electrophoresisFree Flow Electrophoresishttp://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

FHDHydrodynamic FrictionFCFCounter ion FlowParticle Travels UpstreamFFAField Asymmetry Effect

http://www.websters-online-dictionary.org/definitions/Electrophoresis

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Electrophoretic Mobility

Smoluchowski Determined another way to view electrophoretic mobility2Only for Thin double layer

http://en.wikipedia.org/wiki/Marian_Smoluchowski

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ξ

(Zeta Potential)Electric potential in the double layer Potential difference between dispersion medium and cage around particleImportant in stability of particles

http://en.wikipedia.org/wiki/Zeta_potential

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Hückel Correction

Smoluchowski did not correct for Debye lengthLength over which charges are screened3Denoted by κ

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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 membraneElectric FieldFlux of ionsSteady StateFluxes 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 StarchSDS-PAGENative Set up

http://www.aesociety.org/areas/preparative_gel.php

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Agarose and Starch Gels

AgaroseUsed in DNA separation methodsCan be sued in Large protein separations4Can easily be stored for tagging5Starch Also used to separate non-denatured proteins

http://delliss.people.cofc.edu/virtuallabbook/LoadingGel/LoadingGel.html

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SDS-PAGE6

SDSSodium Dodecyl SulfateDenaturantMovement based only on molecular massβ-mercaptoethanol

PAGEPolyacrylamide

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 supportNo DenaturantProtein stays in original conformationProtect from OxidationMovement depends on:Intrinsic Charge7Hydrodynamic Size

http://ccnmtl.columbia.edu/projects/biology/lecture6/index.htm

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Viewing Conditions

Staining depends on type of moleculeView Under UVDNAEthidium BromideGelRedProteinCoomassie Brilliant BlueHorse 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.html6 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.