Electrophoresis

Electrophoresis- Analytical techniques

1. ELECTROPHORESIS
1
EshaBhavinShah
Assistant Professor
Department of Pharmaceutical Chemistry and Quality Assurance
BabariaInstitute of Pharmacy
Bits Edu Campus
Email: [email protected]

2. INTRODUCTION
Inpracticaltermsapositiveelectrode(anode)
andnegativeelectrode(cathode)areplacedina
solutioncontainingions.
Thenvoltageisappliedacrosstheelectrodes,so
soluteionsofdifferentchargeforexample,
anions(negative)andcations(positive)will
movethroughthesolutiontowardstheelectrode
ofoppositecharge.

3. -+
ELECTROPHORESIS
Themigrationofelectricallychargedparticlesorionsin
solutionsduetoanappliedelectricfield.
+ -
3

4. THEORY
Electrohoresisisthemovementofionsunderthe
influenceofelectricalfield.So,separationin
electrophoresisreliesondifferencesinthespeedof
migration(migrationvelocity)ofionsorsolutes.
•Migration Velocity (ν) =μe E
Where, μe=elecrophoreticmobility E=electrical field
strength
Electrophoreticmobilityisafactorthatdetermines
howfastionorsolutemovesinagivenmedium.
•μe = q/ 6Лήr
•where, q=charge of ion ή=viscosity of solution
r=radius of ion

5. Theinstrumentationofcapillaryelectrophoresisisverysimilarto
thatofHPLC.
PowersupplyofEisequivalenttoHPLCpumpandcapillary
equivalenttocolumn.
COMPARISON OF ELECTROPHORETIC AND
CHROMATOGRAPHIC TERMS
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6. ELECTROPHORESIS APPARATUS

7. Principle of Electrophoresis
•Electromigration:
–Ions migrating in electric field
Cationscathode (-ve)
Anions anode (+ve)
7
-+

8. Structure and Properties of Protein
•PROTEINS-polymersofaminoacids
•STRUCTUREofAminoAcids
–aa'shaveacarboxylgroup(-COOH)&aminogroup(-NH2)and
areoftenionizedatphysiologicalpH
•Proteinsareamphotericcompoundsandarethereforeeitherpositively
ornegativelycharged.
8

9. Migration of proteins in electric field
negativelychargedproteinsmovetowardsthe
positivepole
directlyproportionaltotheoverallchargeof
proteins
inverselyproportionaltoproteinsize(molecular
weight)

10. Electropherogram
AnElectropherogramisthe
resultofanelectrophoresis
whichgivesthemovementof
chargedparticlesovertimein
agel,paperoranother
medium.
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11. PROCEDURE FOR
ELECTROPHORESIS

12. 12
Melting the agar…

13. 13
Pouring the gel…

14. Solidification of gel

15. Creation of lanes

16. Formation of lanes

17. Preparation of sample

18. Buffer solution added to the tank
Thisensuresthattheelectriccurrentgoesthroughthewholetankandthatmaintains
thationscanmoveinthesolution

19. samples loaded into wells

20. Electrical current applied to the chamber
Safetycoverisputoverthetopandthecurrentisswitchedon.
Thedyewillmigratethroughthegeltowardthepositiveelectrode,aswilltheDNA
Dependingonhowmuchvoltageisappliedandhowwarmthegelisandsizeandshape
ofmoleculeswilldependonhowfasttheionsmovethroughthegel
Smallerfragmentswillmoveeasiersotheywillbeclosertothepositiveelectrode
Oncethedyehasmovedthroughthegeltothebuffer,theelectricalcurrentisswitched
offandgelisremovedfromthetray

21. VISUALISATION
•Aftertheelectrophoresisiscomplete,themoleculesinthe
gelcanbestainedtomakethemvisible.
•Ethidiumbromide,silver,orcoomassiebluedyemay
beusedforthisprocess.
•Iftheanalytemoleculesfluoresceunderultravioletlight,
aphotographcanbetakenofthegelunderultraviolet
lightingconditions.
•Ifthemoleculestobeseparatedcontainradioactivity
addedforvisibility,anautoradiogramcanberecordedof
thegel.

22. 22
Therearemolecularweightsize
markersavailablethatcontaina
mixtureofmoleculesofknown
sizes.Ifsuchamarkerwasrunon
onelaneinthegelparalleltothe
unknownsamples,thebands
observedcanbecomparedto
thoseoftheunknowninorderto
determinetheirsize.Thedistance
abandtravelsisapproximately
inverselyproportionaltothe
logarithmofthesizeofthe
molecule.

23. Quantification of separated protein band by Densitometer
Densitometerisadevicethatmeasures
thedegreeofdarknessinphotographic
orsemi-transparentmaterial.

24. Types of Electrophoresis:
Zone Electrophoresis:
a)Paper electrophoresis
b)Gel electrophoresis
c)Cellulose acetate electrophoresis
d)Thin layer electrophoresis
Moving boundary electrophoresis:
a)Capillary electrophoresis
b)Isoelectricfocussing
c)Immunoelectrophoresis

25. Zone electrophoresis:
ThetermZoneelectrophoresisisappliedtothosesystemsinwhichionic
mobilitiesarestudiedonstripsofpaper,celluloseacetateoracrylamide.

26. Zone electrophoresis
It involves the migration of the charged particle on the supporting media, paper ,
cellulose acetate membrane, starch gel, polyacrylamide.
The components separated are distributed into discrete zone on the support media.
Supporting media is saturated with buffer solution, small volume of the sample is
applied as narrow band.
On application of potential difference at the ends of a strip ,components migrate at the
rate determined by its electrophoreticmobility.
Advantages:
Useful in biochemical investigations.
Small quantity of sample can be analyzed
Low cost and easy maintenance
Disadvantages:
Unsuitableforaccuratemobilityandisoelectricpointdetermination.
Duetothepresenceofsupportingmedium,technicalcomplicationssuchascapillary
flow,electrosmosis,adsorptionandmolecularsievingareintroduced.

27. PaperElectrophoresis
PaperElectrophoresisisoneofthezoneelectrophoresis.Thisisveryimportantmethod
inalllaboratories.
Paperofgoodqualityshouldcontainatleast95%α-celluloseandshouldhaveonlyavery
slightadsorptioncapacity.
Thistechniqueisusefulfortheseparationofsmallchargedmoleculessuchasamino
acidsandsmallproteins.Astripoffilterpaperismoistenedwithbufferandtheendsofthe
stripareimmersedintobufferreservoirscontainingtheelectrodes.Thesamplesare
spottedinthecenterofthepaper,highvoltageisapplied,andthespotsmigrateaccording
totheircharges.Afterelectrophoresis,theseparatedcomponentscanbedetectedbya
varietyofstainingtechniques,dependingupontheirchemicalidentity.

28. Applications:
Serumanalysisfordiagnosticpurposeisroutinelycarriedaboutbypaper
electrophoresis.
Muscleproteins,eggwhiteproteins,milkproteins&snake,insectvenomanalysis
donebythistechnique.

29. Capillary electrophoresis
Byusingcapillariesasdistinctfrom
flatbeadsystems,itwaspossibleto:
minimizezonespreading,
improveheatdissipation,
shortenseparationtimesand
increaseefficiency.
Applications:
Separationofsteroidsandcomplexdrugmixtureswithin10to15minutes.
analysisofproteinsfoundinserum,urine,CSFandbodyfluids,immunosubstraction
electrophoresis,hemoglobinvariants,lipoproteins,carbohydrate-deficienttransferrin(CDT)
forensicandtherapeuticdrugscreening,andmoleculardiagnostics.

30. Gel electrophoresis:

31. Gelelectrophoresisisamethodforseparationandanalysisofmacromolecules(DNA,
RNAandproteins)andtheirfragments,basedontheirsizeandcharge.
Applications:
Itisusedinclinicalchemistrytoseparateproteinsbychargeand/orsize(IEFagarose,
essentiallysizeindependent).
InbiochemistryandmolecularbiologytoseparateamixedpopulationofDNAandRNA
fragmentsbylength,toestimatethesizeofDNAandRNAfragmentsortoseparateproteins
bycharge.
EstimationofthesizeofDNAmoleculesfollowingrestrictionenzymedigestion,e.g.in
restrictionmappingofclonedDNA.
AnalysisofPCRproducts,e.g.inmoleculargeneticdiagnosisorgeneticfingerprinting
SeparationofrestrictedgenomicDNApriortoSoutherntransfer,orofRNApriorto
Northerntransfer.
Gelelectrophoresisisusedinforensics,molecularbiology,genetics,microbiologyand
biochemistry.
TheresultscanbeanalyzedquantitativelybyvisualizingthegelwithUVlightandagel
imagingdevice.Theimageisrecordedwithacomputeroperatedcamera,andtheintensity
ofthebandorspotofinterestismeasuredandcomparedagainststandardormarkers
loadedonthesamegel.Themeasurementandanalysisaremostlydonewithspecialized
software.
Dependingonthetypeofanalysisbeingperformed,othertechniquesareoften
implementedinconjunctionwiththeresultsofgelelectrophoresis,providingawiderangeof
field-specificapplications.

32. ISOELECTRIC
FOCUSING (IEF)

33. Principle
•Isoelectricfocusingisanelectrophoretic
methodinwhichproteinsareseparatedonthe
basisoftheirpIs.(isoelectricpH)(1-12).
•Itmakesuseofthepropertyoftheproteinthat
theirnetchargesaredeterminedbythepHof
theirlocalenvironment.
•Proteinscarrypositive,negativeorzeronet
electricalcharge,dependingonthepHoftheir
surroundings.

34. •Thenetchargeofanyparticularproteinisthesum
ofallitspositiveandnegativecharges.
•Theyaredeterminedbytheionizableacidicand
basicsidechainsoftheconstituentaminoacids
andprostheticgroupsoftheprotein.
•Ifno.ofacidicgroups>>no.ofbasicgroups,the
pIofthatproteinwillbeatalowpHvalueand
theproteinisclassifiedasacidic.
•Ifno.ofbasicgroups>>no.ofacidicgroups,the
pIofthatproteinwillbeatahighpHvalueand
theproteinisclassifiedasbasic.
•Proteinsshowvariationsinisoelectricpoints,but
pIvaluesusuallyfallinbetweenpH3-12,mostly
betweenpH4-7.

35. •ProteinsarepositivelychargedinsolutionsatpH
valuesbelowtheirpIandnegativelycharged
abovepI.
•So,
pH<<pI---->proteinscarrynetpositivecharge-
migratetowardscathode
pH>>pI---->Proteinscarrynetnegativecharge-
migratetowardsanode
•AtpIaproteinwillnotmoveinanelectric
field.

37. Mechanism
•Whenproteinisplacedinamediumwithlinear
pHgradientandsubjectedtoelectricfield,itwill
initiallymovetowardstheelectrodewithopposite
charge.
•DuringmigrationthroughpHgradient,theprotein
willeitherpickuporloseprotons.
•So,thisresultsinchangeinitsnetchargeand
mobility.
•Eventually,itwillarriveatapointinthepH
gradientequalingtoitspI.
•Therebeinguncharged,itwillstopmigrating
resultsinsharpbands.

38. +pI
acidic
basic
Anode (+)
Cathode (-)
0
0
-
pH

39. •Focusingisthesteadystatemechanismwith
regardtopH.
•ProteinsapproachtheirrespectivepIvaluesat
differingratesbutremainrelativelyfixedatthose
pHvaluesforextendedperiods.
•Thistypeofmotionisincontrasttoconventional
electrophoresisinwhichproteinscontinueto
movethroughthemediumuntilelectricfieldis
removed.
•InIEF,proteinsmigratetotheirsteadystate
positionsfromanywhereinthesystem.
•Thus,sampleapplicationpointisarbitary.

40. Establishing pH gradients
•Establishmentofstable,linearpHgradientsis
accomplishedintwowaysusing
1.Carrierampholytes
2.Acrylamidobuffers

41. 1. Carrier ampholytes
•CA(amphotericelectrolytes)aremixturesofmolecules
containingmultiplealiphaticaminoandcarboxylic
groups.
•Theyaresmall(300-1000Da)multichargedorganic
buffermoleculeswithcloselyspacedpIvaluesandhigh
conductivity.
•CAareincludeddirectlyinIEFgels.
•Inelectricfields,CApartitionintosmoothpHgradient
thatlinearlyincreasefromanodetocathode.
•Thisisthemostcommonandsimplestwaytoobtain
pHgradients.

42. 2. Acrylamidobuffers
•ABarederivativesofacrylamidecontainingboth
reactivedoublebondsandbufferinggroups.
•TheirgeneralstructureisCH2=CH-CO-NH-R,where
Rcontainseitheracarboxylor3
0
aminegroup.
•TheyareincorporatedintoPAGEattimeofcasting.
•Becausethebufferingcompoundsarefixedinplacein
theseparationmedium,thegelsarecalled
“immobilizedpHgradients”orIPGs.
•IPGshavemorestabilitybutarecumbersomeand
expensive.

43. •IEFisahighresolutiontechniquethatcanresolve
proteinsdifferinginpIvalueoflessthan0.05pH
unit.
•Antibodies,antigensandenzymesretaintheir
activitiesduringIEF.
•ThepHrangechosenshouldbecenteredonpIof
theproteinsofinterest.
•WithCA,2%w/visbest
•<1%--unstablepHgradients
•>3%--ampholytesdifficulttoremoveattime
ofstaining
•WithAB,concof3meqisappropriate.

44. Gels for IEF
•ThemostcommoncompositionofgelsforIEF
are
•Polyacrylamidegelswith5%Tand3%C
•Ammoniumpersulfate
•Urea
•0.05%TEMED
•Nowadays,precastIEFminigelsandIPG
sheetsarealsoavailable.

45. Power conditions and resolution
•ThepHgradientandappliedelectricfield
determinetheresolutionofIEFrun.
•pI=sqrtofpHgradient/sqrtofvoltagegradient
•NarrowpHrangeandhighvoltage=better
resolution.
•Highvoltageleadstoshorterruntimebuthasdis
advofgeneratingheat.(jouleeffect)
•Soelectricfieldsoforder100V/cmarepreffered.

46. Protein solubilizationat pI
•SomeproteinstendtoprecipitateoutatpI
values.
•Topreventthis,mostcommonlyUreaisused,
especiallyforthoseproteinsthattendto
aggregateattheirpIvalues.
•Ureaworksbydisruptinghydrogenbonds.(7-
8M).
•Membraneproteinsrequiredetergent
solubilizingagentslikeCHAPSandCHAPSO,
tritonX-100.

47. Detection
•Mostly dyes like amidoblackand coomassive
blue and silver staining are used.
•Autoradiography, liquid scintillation counting
and Flourographycan also be used for
radioactive proteins.

48. Capillary Isoelectric Focusing (CIEF)
It separates analytes according to differences in their isoelectricpoints or pIvalues.
Capillary filled with a solution of carrier ampholytesand sample.
Anodeend of the capillary is placed into an acidic solutionand cathodeend into a
basic solution.
When electric field is applied, due to presence of ampholytecause the pH gradient
into capillary.
Charged sample components then migrate through the capillayuntillthey reach a
region of pH equal to their pI, where they become neutral and therefore, cease to
migrate.
E
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49. Applications of Electrophoresis
1.Forensics
DNAfingerprintofacriminal.
2.MolecularBiologyToseparateandorganizeDNAandRNAby
size
3.GeneticsProvideclearerpictureofDNA,italsohelpsprepare
DNAforcloningandgeneticengineering.
4.MicrobiologyInformationoutabouttheorganisms.Virology:to
helpdiagnosedifferentstrainsofviruses.
5.BiochemistryMappingofcellularcomponents,particularly
proteinsandnucleicacids.
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50. 5.Proteinandpeptidedetermination:
(1)TocheckPurity
(2)PhysicalpropertieslikeMW,pI
(3)Bindingstudies
(4)Identification(CE-MS)
(5)Quantitation
(6)Immunoassays
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6.Determinationofadditivesinfoodanddrug.
Forexample,SeparationofTartazine,Sunsetyellow,amaranth,
indigocarmine
7.SeparationandQuantificationofVitaminsinfruitsand
vegetable.
Forexample,SeparationandquantificationofAscorbicacidin
vegetableandfruits
8.Usedforseparationofenatiomerfromracemicmixture.

51. 51

52. 9.Determinationofminerals,fattyacidsandcarbohydrates.
10.DeterminationofvariousorganicacidslikeHippuricacid,
Oxalicacid,Tartaricacid,Malicacid,Lacticacidinfood.
11.Pharmaceuticalassay
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