The Term Electrophoresis Means Movement of Charged Particles

7/18/2019 The Term Electrophoresis Means Movement of Charged Particles

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The term electrophoresis means movement of charged particles (ions,molecules, macromolecules) in electric eld in liquid medium.Any electrically charged particle dissolved in aqueous solution, when placed to aconstant electric feld, will start to migrate towards the electrode bearing theopposite charge; the speed o the particle movement will be directly proportional to

the applied voltage and particle charge, but inversely proportional to the particlesize. Any molecules that dier in size andor charge can be separated rom eachother in this way. The electrophoretic analysis can in principle be applied to anyparticles that are charged under given e!perimental condition, such as smallcations or anions, organic acids, aminoacids, peptides, saccharides, lipids, proteins,nucleotides, nucleic acids, even the whole subcellular particles or the whole cells. "npractice, however, the by ar commonest sub#ects o electrophoretic separation areproteins and nucleic acids.$lectrophoresis o positively charged particles %cations& is called cataphoresis,while electrophoresis o negatively charged particles %anions& is calledanaphoresis.

General features of electrophoresis The very basic prerequisite o electrophoretic separation is presence o charge onthe separated macromolecules.'hosphate groups in structure o ()A and *)A quite reliably give these substancesnature o polyanions, which in neutral or al+aline p migrate to the positivelycharged electrode %anode&."n case o proteins this issue is somewhat more comple!, because proteins areampholytes, i.e. they bear both positive and negative groups, and the p o themedium %electrophoretic buer& determines whether the resulting net charge oseparated protein is positive or )egative.'roteins are polyanions as well, and hence move rom the cathode %-& to the anode%&.

Electrophoretic techniques: (ierent types o electrophoresis techniques aredesigned depending upon whether it carried out in the presence or absence o asupporting media.

Moving boundary electrophoresisthe electrophoresis is carried in solution, without a supporting media. The sample isdissolved the buer and molecules move to their respective counter chargeelectrodes. /oving boundary electrophoresis is carried out in a 0 shape tube withplatinum electrodes attached to the end o both arms . At the respective ends, tubehas reractometer to measure the change in reractive inde! o the buer duringelectrophoresis due to presence o molecule. 1ample is loaded in the middle o the0 tube and then the apparatus is connected to the e!ternal power supply. 2hargedmolecule moves to the opposite electrode as they passes through thereractometer, a change can be measured. As the desirable molecule passes,sample can be ta+en out rom the apparatus along with the buer.



!isadvantages of Moving "oundary electrophoresis The resolution o thetechnique is very low due to the mi!ing o the sample as well as over3lapping o thesample components. The electrophoresis technique is not good to separate andanalyze the comple! biological sample instead it can be used to study the behavioro the molecule in an electric feld.

#one electrophoresis"n this method, an inert polymeric supporting media is usedbetween the electrodes to separate and analyze the sample. The supporting mediaused in zone electrophoresis are absorbent paper, gel o starch, agar andpolyacrylamide. The presence o supporting media minimizes mi!ing o the sampleand that ma+es the analysis and purifcation o the molecule rom the gel mucheasier than the moving boundary electrophoresis. The gel electrophoresis is thebest e!ample o zone electrophoresis.

Gel electrophoresis:$ertical Gel Electrophoresis: The electrophoresis in this system perormed in adiscontinuous way with buer in the upper and lower tan+ connected by the gelslab. "t has multiple modifcation in the running condition to answer multipleanalytical questions.%ori&ontal Gel Electrophoresis: The electrophoresis in this susyem is perormedin a continous way and the electrophoresis is perormed in the horizontal direction.



'garose gel electrophoresis ('GE)Agarose is a natural linear polymer e!tracted rom seaweed that orms a gel matri!by hydrogen3bonding when heated in a buer and allowed to cool. 4or mostapplications, only a single3component agarose is needed and no polymerizationcatalysts are required.Thereore, agarose gels are simple and rapid to prepare%2hawla, 5667&. They are the most popular medium or the separation o moderateand large3sized nucleic acids and have a wide range o separation but a relativelylow resolving power, since the bands ormed in the gels tend to be uzzy and spreadapart. This is a result o pore size and cannot be largely controlled.

olyacrylamide gel electrophoresis ('GE)'olyacrylamide gels are chemically cross3lin+ed gels ormed by the polymerizationo acrylamide with a cross3lin+ing agent, usually ),)83methylenebisacrylamide. Thereaction is a ree radical polymerization, usually carried out with ammoniumpersulate as the initiator and ),),)8,)83tetramethylethylendiamine %T$/$(& as thecatalyst. Although the gels are generally more di9cult to prepare and handle,involving a longer time or preparation than agarose gels, they have ma#oradvantages over agarose gels. They have a greater resolving power, canaccommodate larger quantities o ()A without signifcant loss in resolution and the()A recovered rom polyacrylamide gels is e!tremely pure %:uilliatt,5665&.



/oreover, the pore size o the polyacrylamide gels can be altered in an easy andcontrollable ashion by changing the concentrations o the two monomers. Anyway,it should be noted that polyacrylamide is a neuroto!in %when unpolymerized&, butwith proper laboratory care it is no more dangerous than various commonly usedchemicals.

'garose gel concentration The percentage o agarose used depends on the size o ragments to be resolved.

The concentration o agarose is reerred to as a percentage o agarose to volume obuer %wv&, and agarose gels are normally in the range o 6.5 to < %1mith,=>><&. The lower the concentration o agarose, the aster the ()A ragmentsmigrate. "n general, i the aim is to separate large ()A ragments, a lowconcentration o agarose should be used, and i the aim is to separate small ()Aragments, a high concentration o agarose is recommended

olyacrylamide gel concentration The choice o acrylamide concentration is critical or optimal separation o themolecules%ames, =>>?&. 2hoosing an appropriate concentration o acrylamide andthe cross3lin+ing agent, methylenebisacrylamide, the pore sized in the gel can becontrolled. @ith increasing the total percentage concentration %T& o monomer

%acrylamide plus cross3lin+er& in the gel, the pore size decreases in a nearly linearrelationship. igher percentage gels %higher T&,with smaller pores, are used toseparate smaller molecules.

$ertical Electrophoresis

. *!*olyacrylamide gel electrophoresis (*!*'GE)+nstrument The schematic diagram o a vertical gel electrophoresis apparatus isgiven in 4igure 55.=. "t has two buer chamber, upper chamber and a lowerchamber. oth chamber are ftted with the platinum electrodes connected to thee!ternal power supply rom a power pac+ which supplies a direct current or (2voltage. The upper and lower tan+ flled with the running buer is connected by the

electrophoresis gel casted in between two glass plates %rectangular and notched&. There are additional accessories needed or casting the polyacrylamide gel such ascomb %to prepare dierent well&, spacer, gel caster etc.



"uer and reagent for electrophoresis The dierent buer and reagents withtheir purpose or vertical gel electrophoresis is as ollows3. -, -, -, -tetramethylethylenediamine (/EME!)it catalyzes theacrylamide polymerization.0. 'mmonium persulfate ('*)it is an initiator or the acrylamidepolymerization.1. /ris%2l it is the component o running and gel casting buer.3. Glycine it is the component o running buer.4. "romophenol blue it is the trac+ing dye to monitor the progress o gel

electrophoresis.5. 2oomassie brilliant blue 6047it is used to stain the polyacrylamide gel.8. *odium dodecyl sulphateit is used to denature and provde negative charge tothe protein.9. 'crylamide monomeric unit used to prepare the gel.. "isacrylamide cross lin+er or polymerization o acrylamide monomer to ormgel.

2asting of the gel: The acrylamide solution %a mi!ture o monomeric acrylamideand a biunctional crosslin+er bisacrylamide & is mi!ed with the T$/$( and A'1 andpoured in between the glass plate ftted into the gel caster. Ammoinum persupatein the presence o T$/$( orms o!ygen ree radicals and induces the

polymerization o acryalide monomer to orm a linear polymer %4igure 55.5&. Theselinear monomers are interconnected by the cross lin+ing with bis3acrylamidemonomer to orm a <3( mesh with pores. The size o pore is controlled by theconcentration o acrylamide and amount o bis3acrylamide in the gel. ") a verticalgel electrophoresis system, we cast two types o gels, stac+ing gel and resolvinggel. 4irst the resolving gel solution is prepared and poured into the gel cassette orpolymerization. A thin layer o organisc solvent %such as butanol or isoproponal& islayered to stop the entry o o!ygen %o!ygen neutralizes the ree radical and slowdown the polymerization& and ma+e the top layer smooth. Ater polymerization o



the resolving gel, a stac+ing gel is poured and comb is ftted into the gel orconstruction o dierent lanes or the samples

6unning of the gel: The sample is prepared in the loading dye containing 1(1, B3mercaptoethanol in glycerol to denature the sample and presence o glycerolacilitates the loading o sample in the well. As the samples are flled vertically thereis a distance drit between the molecules at the top Cs at the bottom in a lane. Thisproblem is ta+en care once the sample run through the stac+ing gel. The p o thestac+ing gel is D.? and at this p, glycine is moving slowly in the ront where as Tris32l is moving ast. As a result, the sample gets sandwiched between glycine3Trisand get stac+ed in the orm o thin band. As the sample enters into the resolving gelwith a p ?.?, the glycine is now charged, it moves ast and now sample runs as pertheir molecular weight %due to 1(1 they have equal negative charge&. Ater trac+ing

dye reaches to the bottom o the gel, gel is ta+en out rom the glass plate with thehelp o a spatula and it is stained with coomassie brilliant blue *5E6 dye. The dyestains protein present on the gel.

*taining:

-ative protein electrophoresis This is a basic arrangement o electrophoresis, in which the proteins retain theiroriginal%native& conormation and oligomeric structure %subunit composition&, charge andbiologic activity,and in mildly al+aline buer migrate to the positively charged electrode. 'garose is

probably the mostcommonly used support or this type o analysis, although similar results can alsobe obtained withcellulose acetate and thin polyacrylamide %as on 4ig. <&. The agarose gel iscustomary also in basicelectrophoresis o nucleic acids, which in this medium separate according to thesize. 4or proteins,however, the pores in agarose are too large or molecular sieving F proteinseparation ta;es places



according to their surface charge density. 'roteins that dier in size, but not incharge density,migrate with the same speed and cannot be resolved %e.g. albumin dimer andmonomer&. Theresolution is limited because there are no further focusing mechanisms. )ormalhuman serum

classically separates to 4 fractions: albumin, <, <0, = and >globulin. The protein separation in agarose sometimes displays an interesting, althoughrather unwantedphenomenon o electroendoosmosis , which maniests as movement o theslowest raction %G3globulins& against the direction o its e!pected electrophoretic mobility, i.e., romthe start towards thecathode %negative electrode&. The reason or this phenomenon is presence onegatively chargedgroups in the agarose gel, around which a cloud o partially mobile positive ionsrom theelectrophoretic buer orms. Hnce voltage is applied, these positively charged ions

start to movetowards cathode and drag the most slowly migrating proteins with them. "n purifedagarose lac+ingthe charged groups and in polyacrylamide the electroendosmosis does not occur.)ative electrophoresis of human serum proteins is one o the basicbiochemicale?aminations, routinely perormed in the clinical chemistry laboratories. "t is alsoused or analysis o isoenzymes, spectra o serum lipoproteins, hemoglobin variants etc.1. @rea 'GE: "n this method, insoluable protein is dissolved in 0rea and samplesseparate based on their chargesubunit mass. A gradient 0ra 'A:$ is used tomonitor the olding states o a protein.

ApplicationI!etermination of Molecular Aeight/olecular weight o a protein can be determined by plotting relative migration *with the log molecular weight o standard protein.!etermination of Bligomeric status of the protein The polyacrylamide gelelectrophoresis can be use to determine the oligomeric status o the protein. Aprotein sample can be run under the denaturating as well as in the native conditionsin two separate gel. The protein o the +nown molecular weight runs on both gelsand a * value is calculated or the standard proteins.*tudying protein foldingCunfolding The urea disrupts electrostatic andhydrophobic interaction between amino acid residues o protein to induce unoldingo the <3( conormation o the protein. "t has been e!tensively used to study theprotein unolding and to identiy dierent structural intermediates in the oldingpathways. "n a typical unolding e!periment, protein is e!posed to the dierentconcentration o urea and then the structural changes in protein can be monitoredby the spectroscopic or gel flitration techniques.urication of the antigen'reparative 1(13'A:$ is routinely been used to puriythe protein %antigen& to generate antibody



roteinprotein interaction There are two approaches in which vertical gelelectrophoresis can be used to study the protein3protein interaction. +n approach +,protein A and is incubated in an invitro reaction to orm the comple! A. )ow theormation o comple! A can be analyzed on a native 'A:$.+n approach 0, the protein A is resolved on the 1(13'A:$ and transerred on thenitrocellulose membrane. The membrane is bloc+ed with the = 1A over3night at

762. )itrocellulose membrane is incubated with the protein over3night at 762./embrane is washed with the buer and probedwith the anti3 antibody ollowed by*' cupled secondary antibody %anti"g:3*'&. lot is developed by the di3aminobenzidine %(A&.!etection of glycoprotein and phosphoprotein 'rotein sample resolved on1(13'A:$ is stained with the dierent reagents to specifcally detect glycoproteinand phosphoprotein.

%ori&ontal gel electrophoresis The electrophoresis in this gel system isperormed in a continuous ashion with both electrodes and gel cassette submersed

within the buer. The electrophoresis chamber has two platinum electrodes placedon the both ends are connected to the e!ternal power supply rom a power pac+which supplies a direct current or (2 voltage. The tan+ flled with the running buerand the gel casted is submerged inside the buer. There are additional accessoriesneeded or casting the agarose gel such as comb %to prepare dierent well&, spacer,gel caster etc.

"uer and reagent for electrophoresis The purpose o each reagents used inhorizontal gel electrophoresis are as ollows3. 'garosepolymeric sugar used to prepare horizontal gel or ()A analysis.0. Ethidium bromide or staining o the agarose gel to visualize the ()A.

1. *ucrose4or preparation o loading dye or horizontal gel.3. /ris%2l The component o the running buer.4. "romophenol blue Trac+ing dye to monitor the progress o the electrophoresis.2asting of the agarose gel The agarose powder is dissolved in a buer %TA$ or

T$& and heated to melt the agarose. ot agarose is poured into the gel cassetteand allowed it to set. A comb can be inserted into the hot agarose to cast the wellor loading the sample. "n ew cases, we can add ethidium bromide within the gel sothat it stains the ()A while electrophoresis.



6unning and staining The gel cassette is placed in the electrophoresis tan+submerged completely and ()A loaded into the well with the help o pipetman and

run with a constant voltage. ()A runs rom negative to positive end and ethidiumbromide %$tr& present in the gel stain the ()A. Hbserving the agarose gel in a 0C3chamber shows the ()A stained with $tr as organe colored Juorescence.

'pplications of %ori&ontal 'garose Gel Electrophoresis

!etermination of si&e of !-' The size o a ()A can be determined bycomparing the size o the +nown ()A molecules. The ()A o +nown sizes areresolved on 6.? agarose along with the un+nown sample!-'rotein +nteraction()A is a negatively charged molecule and it interactwith positively charged protein to orm ()A3protein comple!. The size and thehydrodynamic volume changes when ()A is interacting with protein to orm ()A3

protein comple!.

ElectroelutionAs discussed in previous lecture, protein band present within thepolyacrylamide gel bloc+ is removed by electroelution or urther usage. "n theelectroelution, a gel band is cut rom the 1(13'A:$ and placed in a dialysis bag andsealed rom both ends. The dialysis bag is choosen so that the molecular weight cuto o dialysis membrane should be lower than the protein o interest.

ulsed eld gel electrophoresis is a technique used or the separation olarge deo!yribonucleic acid %()A& molecules by applying to a gel matri! an electricfeld that periodically changes direction.

%istory1tandard gel electrophoresis techniques or separation o ()A molecules providedhuge advantages or molecular biology research. owever, it was unable toseparate very large molecules o ()A eectively. ()A molecules larger than =E356+b migrating through a gel will essentially move together in a size3independentmanner. At 2olumbia 0niversity in =>?7, (avid 2. 1chwartz and 2harles 2antordeveloped a variation on the standard protocol by introducing an alternating voltagegradient to improve the resolution o larger molecules.K=L This technique became+nown as pulsed3feld gel electrophoresis %'4:$&. The development o '4:$



e!panded the range o resolution or ()A ragments by as much as two orders omagnitude.

/heory@hile in general small ragments can fnd their way through the gel matri! moreeasily than large ()A ragments, a threshold length e!ists above <6FE6 +b where all

large ragments will run at the same rate, and appear in a gel as a single largediuse band.

owever, with periodic changing o feld direction, the various lengths o ()A reactto the change at diering rates. That is, larger pieces o ()A will be slower torealign their charge when feld direction is changed, while smaller pieces will bequic+er. Hver the course o time with the consistent changing o directions, eachband will begin to separate more and more even at very large lengths. Thusseparation o very large ()A pieces using '4:$ is made possible.

rocedure The procedure or this technique is relatively similar to perorming a standard gel

electrophoresis e!cept that instead o constantly running the voltage in one

direction, the voltage is periodically switched among three directions; one that runs

through the central a!is o the gel and two that run at an angle o D6 degrees either

side. The pulse times are equal or each direction resulting in a net orward

migration o the ()A. 4or e!tremely large molecules %up to around 5 /b&, switching3

interval ramps can be used that increases the pulse time or each direction over the

course o a number o hoursMta+e, or instance, increasing the pulse linearly rom

=6 seconds at 6 hours to D6 seconds at =? hours.

This procedure ta+es longer than normal gel electrophoresis due to the size o the

ragments being resolved and the act that the ()A does not move in a straight linethrough the gel.

'pplication'4:$ may be used or genotyping or genetic fngerprinting. "t is commonlyconsidered a gold standard in epidemiological studies o pathogenic organisms.1ubtyping has made it easier to discriminate among strains o Listeriamonocytogenes and thus to lin+ environmental or ood isolates with clinicalinections.

Gradient 'GE

The size o pores in polyacrylamide gel can be easily controlled by the compositionandmonomer concentration in the polymerisation mi!ture. "t is even possible to preparea gel in whichpore si&e gradually decreases, i.e. the density o gel continuously increases,towards the anode.

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

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

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

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

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


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

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

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



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



'roteins migrating in such gel during electrophoresis encounter ever increasingresistance o the gelsupport, their migration is progressively retarded and fnally ceases. 1ize o theprotein matters%smaller protein migrates arther&, and so this technique enables separation ofnative proteins or

protein comple?es according to their si&e. *esolution is high because theprotein bands tend to ocusduring their separationI the protein molecules that are Nbehind8 are still in a thinnergel, hence moveaster and tend to Ncatch up8 the protein molecules that are more Nahead8 and somore retarded by thegel resistance.

2apillary electrophoresis

All the e!amples o gel electrophoresis e!plained above are perormed in rods or%much more

oten& slabs o cast gel. "n capillary electrophoresis the technical arrangement isdierentI theseparation ta+es place in very thin %internal diameter =63=66 Om& and airly long%563566 cm&capillary, which e!tends with its ends to the chambers containing electrophoreticbuer and theelectrodes. The actual electrophoretic separation can be based on any o theaorementioned principles,i.e., it can proceed as ree or gel electrophoresis, isoelectric ocusing etc. A portiono capillarytypically serves as window or detection o separating components that can bebased on photometryor lasere?cited Duorescence. )o permanent electrophoreogram is obtained;rather, the wholearrangement resembles chromatographic analysers.

The capillary electrophoresis oers two critical advantages. The frst one ispossibility o ecient removal of electric currentgenerated heat #ust because o smalldiameter o the capillary.2onsequently, high voltage can be applied that shortens time o the whole analysis%sometimes evenless than = minute per sample&. The thin capillary also solves the problem oconvection Jow in reeelectrophoresis. The other big advantage is easy automation. The routineelectrophoresis o serumproteins can be completely automated in this way. Another good e!ample is the*angerFs techniqueof !-' sequencing, also based on electrophoresis. *eplacement o the originallaborious hand3madecast sequencing gels with the capillary electrophoresis was one o the criticaladvances that enabled



development o ecient automated sequencers, necessary or the ormidabletas+ o human genomesequencing.

+soelectric focusing

The technique called isoelectric ocusing %"$4& separates proteins purely ande?clusivelyaccording to their charge, and allows determination o isoelectric point o aprotein. This isachieved by electrophoretic separation o proteins in gradient of p%, resultingrom a comple?mi?ture (4777) of organic ampholytes %amphoteric polyaminocarbo!ylicacids&, each withdierent isoelectric point. " voltage is applied, the ampholytes position in the gelaccording to theirisoelectric points, which produces the continuous p gradient. The sample proteins,then, in this

environment migrate to the anode or cathode until each protein fnds a place wherethe local p%corresponds to its isoelectric point, and stays there because at pPp" the netprotein charge is zero.

The bands display a tendency to focusing because once a protein moleculediuses out rom the placewhere pPp", it regains charge and is orced by the electric feld to move bac+ tothe band center %thatis why it is called isoelectric focusing&. This technique can resolve proteins whosep"s dier by as littleas 6.65 p units.

2D Electrophoresis

"n the two3dimensional %5(& electrophoresis a mi!ture o proteins is frst sub#ectedtoseparation according to their charge by isoelectric ocusing, and then in the seconddimensionaccording to their size in the presence o 1(1 %4ig. 7&. 2ombination o twoseparations, one based onp" and the other one on molecular weight, oers resolution that cannot compare toany o the N=(8techniques F one sample on a single 5( gel can yield several thousands o proteinspots.



rinciple of 0! electrophoresis. eft: isoelectric ocusing in rods o thinpolyacrylamide, proteinsmigrate in p gradient and ma+e separated zones according to their isoelectricpoints. 6ight: ollowing the "$4 the gel rod with protein bands is equilibrated with1(1 and placed upon a slab o a denser polyacrylamide,protein zones rom the frstdimension now undergo second separation according to the molecular weight opolypeptide chains. %According to materials o 'harmacia 4ine 2hemicals, 0ppsala,1weden, =>?<&





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The Term Electrophoresis Means Movement of Charged Particles