Gel Electrophoresis • Polyacrylamide • Agarose

Electrophoresis Overview

ดร. เลอสรร ธนสุกาญจน์ ภาควิชาชีวเคมี คณะวิทยาศาสตร์ จุฬาลงกรณ์มหาวิทยาลัย

• slip plane (shear plane), whose potential = electrokinetic (zeta) potential • Gouy layer (Gouy-Chapman layer or diffused layer) = layer of counterions starting from the outer Helmholtz plane • Stern layer = uncharged region between the surface and the locus of hydrated counterions • outer Helmholtz plane, whose potential = diffused layer potential (Stern potential) = potential at the beginning of the diffused layer • outer Helmholtz layer • inner Helmholtz plane, distance = ß from the surface • inner Helmholtz layer • Surface with adsorbed charged at distance = 0 charge-free Stern layer

• Electrophoresis • movement of charged colloidal particles or polyelectrolytes, immersed in a liquid, under the influence of an external electric field • Electro-osmosis • motion of a liquid through an immobilized set of particles, a porous plug, a capillary, or a membrane, in response to an applied electric field. • etc.

• competition between motion along the electric field and diffusion in 3 dimensions --> spreading of a protein band • terminal velocity reached very quickly after the electric field is switched ON --> net force in the direction of motion is zero and the particle moves at a constant speed in the forward direction

• Dielectrophoresis = the motion of uncharged particles due to induced polarization from nonuniform electric ﬁelds • Electrowetting = changes in wettability of an electrolyte droplet on a surface by an electric ﬁeld

−1 −1 • Electrophoretic velocity, ν e m i s cm i s

ν • Electrophoretic mobility, u = e m2V−1s−1 cm2V−1s−1 e E

• Moving-boundary Electrophoresis • Paper & Cellulose Acetate Electrophoresis • Gel Electrophoresis • polyacrylamide • agarose

http://www.funsci.com/fun3_en/exper1/exper1.htm

• e.g. in 2D ﬁngerprinting of low mw substances

http://physicalchemistryresources.com/Book3_sections/E_High%20Voltage%20Electrophoresis_html_web.htm

• acetate replaces hydroxyl groups • not adsorb on proteins •

http://www.ou.edu/journals/dis/DIS83/Teach83/ThompsonT1/Thompson.htm

1.small, charged 2.larger, more charged 3.smaller, less charged Middle: same mass different charges Right: different mass same charge/mass

Van Holde, K.E. 2006. Principles of Physical Biochemistry, 2nd ed. p. 253 • cross-linked gel --> pore

• selection by size 0 logUri = logUri − kiC • Ferguson plot

• Uri = distance moved / distance tracking dye moved 0 • U ri = Ur of ith molecule at 0 gel concentration • C = gel concentration

• partially hydrolyzed tapioca starch heated to 70ºC and cooled down to form a gel • pore size depends on • starch concentration • extent of hydrolysis • retardation coeﬃcient (r) depends on • molecular size • Problems • negative side chains make starch an ion exchanger

• synthetic, uniform gel • initiated by • ammonium persulfate + TEMED • riboﬂavin + TEMED + UV • termination by oxygen

อ้างถึงในสุกัญญา สุนทรส 2549 อีเล็กโทรโฟรีซิส • total acrylamide (%T) = conc. of acrylamide + bis acrylamide in gm per cent (w/v) • % cross linker = % bis acrylamide/total acrylamide

• need to recirculate buﬀer in longer runs

• Tube gels vs Slab gels • nowadays slab gels are routinely cast • tube gels are used in rare instances • Denaturating vs Non-denaturating buﬀer • Continuous vs Discontinuous buﬀer system

• older method • easier to cast • concurrent run of varying gel concentrations • diﬃcult to cool

http://bioeraindia.com/product/Tube-Gel-Electrophoresis-Unit/1178.aspx

• easy to keep the same condition for all lanes • easier to cool than tube gels

http://www.medicine.cmu.ac.th/research/equip/Vertical.html

• Native gel <= use non- denaturating buffer • Denaturating gels <= add http://renalfellow.blogspot.com/2012/07/urea-and-hyponatremia.html denaturant(s) to buffer • 9M urea (does not affect charges on proteins) • 6M guanidine hydrochloride

http://www.scbt.com/datasheet-202637-guanidine-hydrochloride.html

Copyright © 2009 by Lerson Tanasugarn. All rights reserved. 20 Discontinuous (Disc) Gel Eletrophoresis pH 8.3 buffer with glycine (pI ~ 6) pH 6.8 stacking gel buffer & sample buffer containing Cl- pH 8.9 running (separating) buffer containing glycine pH 8.3 buffer with glycine

http://ww2.chemistry.gatech.edu/~lw26/bCourse_Information/4581/techniques/gel_elect/page_protein.html A zone of low anion concentration in the stacking gel produces higher electric ﬁeld, which accelerates the proteins so that they enter the separating gel as a narrow band at the boundary between the leading Cl- and the trailing glycine ions.

Copyright © 2009 by Lerson Tanasugarn. All rights reserved. 21 In the stacking gel, as power is turned on • at pH 6.8 • glycine net charge is about -0.001 • -0.99996 from dissociating alpha carboxyl group • +0.99895 from protonated alpha amino group • chloride net charge is -1 • As the power is turned on • a narrow zone of low conductance is created in the stacking gel • Cl- moves quickly downward towards the + terminal • glycine moves slowly towards the + terminal • a very high electric ﬁeld is created in the stacking gel • across a very high resistance in series with a very low resistance • all proteins are accelerated through a low % polyacrylamide gel, reaching the running gel at the same time (they cannot outrun the chloride ion front) • concentrating eﬀect on the protein sample --> tight bands

• glycine is now negatively charged due to high pH of the running gel • glycine races past the proteins • proteins are now in a much more relaxed, uniform electric ﬁeld

• SDS binds protein at about 1 SDS : 3 peptide bonds • peptide is now rod shape, net charge swamps out native charges and is proportional to the number of peptide bonds, hence the molecular weight • S-S bonds need to be cleaved • plot low mw vs Rf

Weber & Osborn, 1969 อ้างถึงในสุกัญญา สุนทรส 2549 อีเล็กโทรโฟรีซิส

• Acrylamide DNA gels • good for DNA a few hundred bases in length • polymerize & cross-link inside the slab or tubes • usually run vertically • Agarose DNA gels • good for DNA a few hundred to tens of thousands bases long • allow boiled agarose solution to solidify in the slab into a translucent gel • usually run horizontally

• Agar = complex mixture of 2-3 polysaccharides with the same backbone structure but with variable degrees of charged groups (mostly acidic) • agarose • agaropectin • Agarose melts at >85ºC, gels at 32-39ºC with brittle, crunchy texture • uncharged -> low adsorption and electroendosmosis • serves as an anticonvection agent

• Intercalating dyes • Ethidium bromide • Acridine orange • Newer, less toxic stains • Visualizable under UV • Log base pairs is linear with Rf

Northern Blot hybridization probe

Western Blot

Southern Blot 1979, protein immunoblot 1975

Southwestern Blot protein SDS PAGE blotted onto nitrocellulose then +

• PAGE voltage about 100-150 volt, electric ﬁeld limited by heat dissipation • capillary --> higher surface area to mass --> easier to cool --> possible to use higher electric ﬁeld --> faster speed of migration • Many types of capillary electrophoresis • free solution capillary electrophoresis, FSCE • gel capillary electrophoresis; capillary electrophoresis with polymer matrices (for SDS CE)

• certain surfaces, e.g. silanols, can dissociate and become ionized at a certain pH range, the layer of counterions can move water molecules under an electric ﬁeld • no problem in capillary electrophoresis since the movement is uniform throughout the column and the ﬂow is laminar • Stacking, i.e. concentrating the protein sample, can be achieved by lowering the ionic strength of the sample to about 1/10 of that in the capillary.

• real-time monitoring of electrophoresis in-progress • much faster than conventional electrophoresis • less sample needed • less reagent & solvent --> more economical • can be automated