ELECTROPHORESI STECHNI QUES

Electrophoresisisal aborator
yt echnique thatuses an el ectri
cf ieldt o
separateandanal yzechargedparticl
es,suchasDNAf ragments,proteins,andot her
macr omol ecules,based on theirsize,charge,ormobi l
ityin a gelorsol ut
ion.
Electr
ophor esisiswidelyemployedi nmolecularbi
ology,bi
ochemi stry
,genet i
cs,and
cl
inicaldiagnost i
cs.Her
ear esomecommonel ectr
ophoresistechniques:

1.AgaroseGelEl ectrophoresis:
Agarosegelel ectr
ophor esi
si sawidelyusedlaborat
orytechniquei nbiochemi
stry
andmol ecularbiol
ogyf ort hesepar
ati
onandanal ysi
sofmacr omoleculessuchas
DNA,RNA,and pr otei
ns based on t hei
rsize and charge.Thi st echni
que i
s
fundamentalf ort asks l i
ke DNA f r
agmentsi zi
ng,DNA pur if
icati
on,and the
assessment of DNA or RNA i nt
egrity.Here'
s an overview of agar ose gel
elect
rophoresi
s:

Pri
nciple:
Agarosegelel ectr
ophoresi
st akesadv ant
ageofthefactthatchargedmolecules
(e.
g.,DNAandRNA)wi llmovet hroughagelmatri
xwhenanel ect
ri
cfiel
disappl
ied.
Ther ateofmov ementdependsont hesizeandchargeoft hemol ecul
es,with
smallermoleculesmigr
atingfasterthr
oughthegel
thanl
argerones.

KeyComponent s:
1.Agar oseGel :Agar ose,der i
vedf rom seaweed,i st hegelmat rixusedi nt his
technique.Theconcent rati
onofagar osecanbeadj ustedt ocr eategelswi t
h
varyingpor esizest osepar atemol ecul esofdi f
ferentsi zesef f
ect iv
ely.
2.El ectrophor esisAppar atus:Thi sincl udesapowersuppl y,anel ect r
ophoresis
chamber ,andel ectrodes.Thegeli spl acedi nthechamber ,andanel ectri
cal
cur r
enti sappliedacr ossit,causingchar gedmol eculest omi gr at e.
3.Buf f
er :Anel ectr
oly t
ebuf fersolutionsur roundst hegel ,mai nt ainingast able
pHandpr ovidi
ngi onst oconductt heel ectri
ccur rent.
4.Loadi ngDy e:At rackingdy econt ai ningcol oredmol ecules( e.g., bromophenol
blue)i sof t
enmi xedwi tht hesampl ebef oreloadi ngi tintot hegel .Thisdy e
helps moni torthe pr ogress ofel ectr ophoresis and v isualize t he sampl e
mi gration.

Pr
ocedur e:
1.GelPr eparation:Agar ose powderi s mi xed with buf fer,heat ed unt i
li t
dissolves,andt henpour edi nt
oacast ingt r
ayt osoli
di f
y.Onceset ,thegeli s
submer gedinel ectrophor esisbuff erint heelectr
ophor esi
schamber .
2.Sampl eLoadi ng:Thesampl e,typicall
ymi xedwi t
hal oadingdy e,iscar ef
ull
y
l
oadedi ntowel lsatoneendoft hegel usingami cropipette.
3.El ectrophoresi s:Anel ectriccur renti sappl i
edtot hegel ,causingchar ged
mol eculest omov et hrought hegelmat ri
x.Thesmal l
ert hemol ecule,the
fasteritmi grat es.DNAorRNAf ragment saresepar atedbysi zedur ingt hi
s
process.
4.Vi sualizati
on:Af terelect rophoresi s,thegeli sstai
nedwi t
hady e,suchas
ethidi
um br omi deorSYBRGr een,whi chbi ndstonucl eicaci ds.UVl ightor
bluelightisusedt ovisualizethesepar atedbandsunderageldocument ati
on
system.

Appl
icat
ions:
 DNAF ragmentSi zi
ng:Agarosegelel
ectr
ophoresi
sisusedt
oesti
matethe
si
zesofDNA f ragments,such asthose pr
oduced byPCR orr
est
ri
cti
on
enzymedigest
ion.
1
  Pur
if
icat
ion:I
tcanbeusedt
osepar
ateDNAorRNAf
ragment
sofi
nter
est
f
rom ami
xtur
efordownst
ream appl
i
cat
ions.
 AssessmentofPur
it
y:Thet
echni
quecanassesst
hepur
it
yandi
ntegr
it
yof
DNAorRNAsampl
esbyr
eveal
i
ngi
mpur
it
ies,
degr
adat
ion,
orcont
ami
nat
ion.
 Genot
ypi
ng:I
t'
sused i
ngenot
ypi
ng exper
iment
stoi
dent
if
yspeci
fi
cDNA
sequencev
ari
ati
ons,
suchassi
ngl
enucl
eot
idepol
ymor
phi
sms(
SNPs)
.
 Qual
it
yCont
rol
:Agar
osegel
elect
rophor
esi
sisar
out
inequal
i
tycont
rol
stepi
n
mol ecularbiol
ogyexper iments.
2.Poly acrylamideGelElectrophoresis(PAGE)
Pol yacry
lamide GelEl ect
rophoresi
s( PAGE)i s a widely used laborator
y
techniquei nbiochemistryandmol ecul
arbiologyfortheseparati
onandanal y
sisof
biomol ecules,pri
maril
ypr oteinsandnucl ei
cacids( DNAandRNA) .Itisespeciall
y
valuablef orresolvi
ngcompl exmi xtur
esofmol eculesbasedonsi ze,charge,and
conformat ion.Hereisanov erviewofpolyacry
lamidegelel
ectrophoresis:

Pri
nciple:
PAGEi sbasedont heprinci
plethatchar
gedbiomoleculeswil
lmigr
atethr
ougha
porouspol y
acr
ylamidegelmat ri
xwhensubject
edtoanel ect
ri
cfi
eld.Therateof
migrati
ondependsont hesizeandchargeofthemolecules,wi
thsmall
ermolecul
es
mov i
ngf ast
ert
hanlargerones.

KeyComponent s:
1.Pol yacr yl
ami deGel :Unli
keagar osegel susedi nagar osegelel ectrophor esi
s,
polyacrylami degel sar e sy nt
heticand of ferhi gherr esoluti
on f orsmal l
er
biomol ecules,suchaspr oteinsandshor tDNAf ragment s.
2.El ectrophor esisAppar atus:Thisi ncludesapowersuppl y,anelect rophoresis
chamber ,andel ect rodes.Thegeli spl acedi nt hechamber ,andanel ectr
ic
currentisappl iedacr ossi t
.
3.Buf fer:Anel ectr
oly tebuf f
ersol utionsur roundst hegel,mai nt
ainingast able
pHandpr ovidi
ngionst oconductt heel ectri
ccur rent.
4.Loadi ngBuf fer:Al oadingbuf f
erisaddedt othesampl esbef or
el oadi ngthem
ontot hegel .Thisbuf f
ercont ainst rackingdy es( e.
g.,bromophenolbl ue)t o
moni torthepr ogressofel ectr
ophor esisanddenat uri
ngagent s( e.g.,SDSf or
proteins)tol i
nearizemol eculesandensur euniform mi grati
on.

Pr
ocedur e:
1.GelPr epar ati
on:Apol yacrylamidegeli spr eparedbymi xi
ngacr ylami deand
bis-acr yl
ami demonomer s,apol ymer izati
oni nit
iator,andacr osslinker.This
mi xturei s pour ed into a cast ing t ray and al l
owed t o polymer ize.The
concent rati
onofacr ylami de/bis-acrylamidedet er
mi nest hegel '
spor esize
andt husi t
ssepar ati
onr ange.
2.Sampl eLoadi ng:Sampl esar emi xed wi thal oading bufferand car efull
y
l
oadedi ntowel lsatoneendoft hegel .
3.El ect rophor esis:Anel ectriccur renti sappl iedt ot hegel,causi ngchar ged
biomol eculest omi gratet hrought hepol y acr yl
ami demat ri
xbasedont heir
sizeandchar ge.Smal lermol eculesmov efast er.
4.Vi sual izati
on:Af t
erelect rophoresi s,thegeli sst ai
nedwi t
hspeci f
icdy esthat
bindt ot hebi omol eculesofi nterest.Forpr otei
ns,Coomassi eBl ueorsi l
ver
stainingi scommonl yused.ForDNAorRNA, ethidi
um br omideorSYBRGr een
i
sof tenempl oy ed.Thesepar at
edbandsar ev i
suali
zedunderUVl ightorbl ue
l
ight .

Appl
icat
ions:
 Pr otei
nSepar
ati
on:PAGEi
susedt
osepar
ateandanal
yzepr
otei
nsbasedon
2
 theirmol ecularwei ght,conf ormat i
on,orchar ge.Itiscommonl yusedf or
proteinpur i
fi
cati
on,qual i
tycont r
ol,andpr oteincharacteri
zation.
 DNAandRNAAnal ysis:PAGEcansepar ateDNAandRNAmol ecul
esbysi ze,
all
owi ngt heassessmentoff r
agmentl engthsandpur ity
.Iti susedi nDNA
sequenci ng,RNAanal y si
s,andgenot ypi
ng.
 Prot ei
n-DNA I nteractions: PAGE can be used t o st udy pr otei
n-DNA
i
nt eracti
ons,suchasDNA- bindi
ngpr otei
nsorpr otei
n-DNAcompl exes.
 Mol ecularBi ol
ogy Techni ques:PAGE i s empl oyed inv arious molecular
biologytechniques,includingSout hernblot t
ing(DNA) ,Northernblot
ting(RNA),
andWest ernblott
ing( protei
ns).
 Qual it
yCont rol
:Itisusedt oassesst hequal i
tyandi ntegri
tyofDNA,RNA,or
proteinsampl esinresear chandcl ini
caldi agnosti
cs.

3.Sodium Dodecy lSulfat

ePol y
acr ylamideGelEl ect
rophoresis
Sodium Dodecy lSulf
ate Pol yacryl
amide GelEl ectrophor esis,commonl y
knownasSDS- PAGE,i saspeci al
izedf orm ofpolyacry
lami degelel ect
rophoresi
s
usedf orthesepar ati
onofpr oteins.SDS- PAGEi sapower fult echniquet hatall
ows
forthedeterminati
onoft hemol ecularweightandpur i
tyofpr oteins.Itisast andar
d
toolinbiochemistry,molecul
arbi ology,andpr ot
eomics.Her e'sanov ervi
ewofSDS-
PAGE:

Principle:
Thef undament alpri
nci
pleofSDS- PAGEi st oseparat
eprotei
nsbasedpr i
maril
yon
theirmol ecularwei ght(si
ze).Thisi s achieved bydenatur
ing the prot
eins and
applyinganegat i
vel
ychargeddetergentcalledSodium DodecylSulf
ate(SDS),whi ch
i
mpar tsauni form negat
ivecharget othepr otei
ns,maskingtheirinher
entchar ge.
Consequent ly
,t heprotei
nspr i
mari
lymi grat
et hroughthepoly
acrylamidegelbased
ont heirsi
zerat hert
hantheirchar
ge.

KeyComponent s:
1.Pol yacr yl
ami deGel:Simi l
art ot raditi
onalpol yacr y l
amidegelel ect
rophoresis,
SDS- PAGE uses a pol yacr ylami de gelas t he separ ati
on mat ri
x.The gel
concent rationi sadjust ed dependi ng ont hesi zer angeofpr ot
einsbei ng
analyzed.
2.SDSSampl eBuf fer:Sampl escont aini
ngpr ot einsar emi xedwi thanSDS-
containingsampl ebuf fer, whichal sot ypicall
yi ncl udesar educingagent(e.g.,
β-mer capt oethanolordi thiothreitol)tobreakdi sulfidebondsanddenat urethe
proteins.
3.Runni ngBuf fer
:Anel ectrophor esisbuf f
ersur roundst hegel andprovidesions
forconduct ingtheelectriccur rentdur ingelectr ophor esis.
4.El ectrophor esisAppar at us:Thi sincludesapowersuppl y,anelectr
ophoresis
chamber ,andel ect
rodes, similart ostandar dgel electr
ophor esi
ssetups.

Pr
ocedure:
1.GelPr eparation:Apol yacry l
ami degelispr eparedwi tht
hedesiredper centage
ofacr yl
amide and bi s-acr yl
ami de to create a por e si
ze suit
ablef ort he
separati
onoft het argetpr oteinr ange.
2.Sampl ePrepar ati
on:Pr ot einsampl esaremi xedwi thSDSsampl ebuf f
erand
heated.Thisdenat urest hepr oteins,breaksdi sul
fidebonds,andcoat sthem
withSDS,pr ov i
dingauni form negat i
vechar ge.Thi sstepalsolineari
zest he
protei
ns,al
lowi ngthem t omi gratethrought hegel basedsolel
yonsi ze.
3.Loadi ng:Denat uredpr oteinsampl esareloadedi ntowellsatthetopoft hegel.
Molecularwei ghtmar kersorl adder sareof tenloadedi nseparatewel lsfor
compar i
son.
3
 4.Elect
rophor esis:An elect ri
c curr
enti s appli
ed tot he gel,causing the
negativ
elychar gedSDS- coatedprotei
nst omigrat
et hroughthegelmat ri
x.
Smal l
erproteinsmov efast erandtr
avelfar t
hert
hroughthegel.
5.Visuali
zation: Af t
er electrophor
esis,t he gel ist ypical
ly st
ained wi t
h
Coomassi eBl ueorsi l
verst aintov i
sual izet
hesepar ated pr
otein bands.
Alter
nativ
el y
,West ernbl
ot ti
ngcanbeper f ormedforspecifi
cprot
eindetecti
on
usingantibodies.

Appl
icati
ons:
 Pr ot ei
nSi zeDet erminat i
on:SDS- PAGEi susedt oest i
mat ethemol ecul
ar
weightofpr oteins bycompar i
ng theirmi grati
on distances to mol ecul
ar
weightmar kers.
 Pr ot ei
nPur it
yAssessment :Ithel
psi nassessingthepur it
yofpr ot
einsampl es
andi dentif
yingthepr esenceofcont aminantsorot herprotei
ns.
 Pr ot ei
nSepar ation:SDS- PAGEcansepar atecompl exmixturesofpr ot
einsfor
furtheranalysisorpur i
ficati
on.
 Qu alit
yCont r
ol:Iti swi delyusedi nqual i
tycont rolproceduresforpr otei
n-
basedpr oducts,suchasphar maceut i
calsandbi ot
echnol ogyproducts.
 Pr ot ei
n Char acterizati
on:SDS- PAGE i s often a pr el
iminary step int he
char acteri
zati
onofpur ifiedpr ot
einsbef or
emor eadv ancedt echniquesl i
ke
massspect romet ryorNMRanal ysi
s.

4.Capill
aryElectr
ophor esi
s( CE)
Capil
laryElectrophoresi s( CE)isapower f
ulanalyti
calt echniqueusedf orthe
separati
onandanal ysisofions, smallmolecul
es,andbiomol eculesinanar row-bore
capi
ll
aryf il
led wi t
h an el ect rol
yte sol
uti
on.Iti s based on t he principles of
el
ectrophoresisandof fersadv antagessuchashighresolution, speed,andv ersati
l
ity
.
CE is wi dely used i nv arious f i
elds,incl
uding biochemi stry,chemi stry,and
biot
echnology.Her e'
sanov erv iewofcapill
aryel
ectr
ophor esis:

Princi
ple:
CEr eli
esont hemigrati
onofchar gedanaly
testhroughacapi l
lar
yfil
l
edwi than
electr
olyt
esol
uti
onwhenanel ectri
cfiel
disappli
ed.Themi gr
ati
onratedependson
thechargeandsizeoftheanal
ytes,
aswel l
asthebuffercomposi
ti
onandpH.

KeyComponent s:
1.Capi llaryTube:Anar row- bor ecapi
ll
aryt ube( ty
pical
lymadeoff usedsili
ca)
servesast hesepar ationcol umn.It
ssmal ldiamet erresult
sinhi gheffi
ciency
andr esolut ion.
2.El ectrolyteBuf fer:Anel ectrolyt
esoluti
onf ill
st hecapill
arytubeandser vesas
themedi um t hroughwhi chi onsormol eculesmi grat
e.Thebuf fercomposi t
ion
canbeadj ustedt oopt imi zesepar at
ioncondi tions.
3.El ectrodes:El ectrodesatbot hendsoft hecapi l
lar
ycr eat
et heel ect
ri
cf i
eld
requiredf ormi grati
on.
4.Det ect or:Var iousdet ect ors,suchasUV- visi
bleabsor bance,fluorescence,or
conduct ivitydet ectors, canbeusedt omoni t
oranal yt
emi grati
onandquant if
y
separat edcomponent s.

Pr
ocedur e:
1.Sampl eIntr
oduct i
on:Thesamplecontai
ninganalytesisintroducedintothe
capil
lary
.Thi s can be done usi
ng t
echniques li
ke pressurei nj
ecti
on or
el
ectrokinet
ici
njecti
on.
2.El ectr
ophoresis:An elect
ri
cfieldisappli
ed acrossthe capi l
lary
,causing
analytestomi gratethr
oughtheelect
rol
ytesoluti
on.Ther ateofmi grat
ion
4
 dependsont heanal yte'scharge,size,andint
eracti
onswi tht
hebuffer
.
3.Detection:Asanal ytesmi grat
et hrought hecapil
l
ary ,
theypassbythedetect
or,
andt heirconcentrationorabsor bancei smeasured.Thisallowsthecreati
on
ofelectropherograms, whichrepresenttheseparationpatter
n.
4.DataAnal ysis:Dataobt ai
nedf r
om t hedetectorareanalyzedtodeter
minethe
i
dent i
tyandquant ityofsepar atedanal yt
es.

Appli
cat i
ons:
Capil
l
ar yel ect r
ophor esishasawi der angeofappl icat i
ons,i
ncluding:
 DNASe quenci ng:CEi susedf orDNAsequenci ng,allowingt hedet ermi nation
ofnucl eot i
desequencesef fi
cient lyandaccur ately
.
 Pr ot ein Anal ysis:Iti s empl oyed f orpr otein characterizat
ion,i ncl uding
deter mi ningpur ity
,molecul arwei ght,andpost -t
ransl
at i
onal modifications.
 Ph ar maceut i
calAnal ysis:CEi susedi nphar maceut icalresearchandqual it
y
cont rol fordruganal ysisandi mpur i
typrofil
ing.
 E nv ironment alAnal ysi
s:I tcandet ectandquant i
fyenv ir
onment alpol l
ut ants,
i
ncl udi ngheav ymet al
sandor gani ccompounds.
 F oodandBev er ageAnal ysis:CEi susedf orf oodsaf etyandqual i
tycont rol,
suchasdet erminingthecont entofaddi ti
vesorcont ami nants.
 Cl i
ni calDi agnost i
cs:CEi sut ili
zedi ncl i
nicall aborat
or i
esf oranal yzi
ngser um
prot eins, hemogl obinvariants, andcl ini
cal biomar kers.
 Ch i
r alSepar ations:CE cansepar ateenant iomer s( mirr
or-imagei somer s),
whi chi sv aluableinphar maceut i
calsandagr ochemi cals.

5.Isoelectr
icFocusing(IEF)
Isoel
ectricFocusing(IEF)i sahi gh-r
esoluti
onelectr
ophor
esistechni
queusedt o
separateand anal y
zepr otei
nsand ot heramphot er
icmolecul
esbased on t hei
r
i
soelectri
c point
s( pI).Itis a power f
ulmet hod forchar
acter
izi
ng prot
eins and
peptidesaccordi
ngt otheirchargepropert
ies.Her
e'sanover
viewofIEF:

Princi
ple:
Thepr imarypr i
nci
pleofI EFistosepar atemol eculesbasedontheiri
soelect
ric
points(pI
),whichisthepHatwhi chamol eculecarr
iesnonetel
ect
ri
ccharge.Inan
el
ect r
icfi
eld,
protei
nswi l
lmi gr
atetothei
rpI ,wheret
heybecomefocusedandremai n
stati
onary.ThepH gr adientestabl
ishedwi thi
nt heseparati
onmedium al
lowsf or
separati
onbasedonpIv alues.

KeyComponent s:
1.GelorCapi ll
aryMedi um:Thesepar at
ion medi um i st ypi
callyagelora
capil
laryt ube fil
led wi t
h an amphol yt
e mi xture,whi ch establi
shes a pH
gradiental ongi t
sl ength.Amphol ytesar emol eculest hatcanactasbot h
acidsandbases, helpingtomai ntai
nast abl epHgr adient.
2.El ectrodes:Anel ectri
cf i
eldisappli
edacr ossthegelorcapi l
larybyusingt wo
electr
odes,oneateachend.Thi select r
icf i
elddrivesthechar gedmol ecules
towar dtheirrespectiv
epIv alues.
3.Sampl e:Thepr otei
nsampl eisintr
oducedatoneendoft hegelorcapi l
l
ar y
.
Proteinswi llmigrat
ewi t
hinthepHgr adientuntiltheyreacht heirpI,atwhi ch
pointtheyst opmov ing.

Pr
ocedure:
1.GelPr eparati
on:AgelwithapHgradienti
sprepar
edusi
ngampholyt
es.The
pHgradientcanbel
inearornonl
i
near
,dependi
ngonthespeci
fi
cappl
i
cati
on.
5
 2.Sampl eLoadi ng:Thepr ot
einsampl eisloadedont othegelorcapill
aryatthe
acidicend.
3.Electrophoresis:Anelectr
icfi
eldisapplied,causi
ngproteinst
omi gratewithi
n
the pH gr adient.Protei
nswi llmov e unti
lt heyreach the pH region t
hat
mat chestheirpI.Atthi
spoi nt
,theirnetchargebecomesneut r
al,
andt heystop
mi grati
ng.
4.Det ecti
on:Af terelect
rophoresis,thesepar at
edpr otei
nscanbev i
sual
ized
usingv ari
ousst aini
ngmet hodsordet ectedbyspecifi
cassays,dependingon
theexper i
ment algoal.

Appl
icat
ions:
 Pr oteinSepar ation:I EFisusedt osepar atepr oteinsbasedont heirpIv alues,
al
lowingf ort hei sol ati
onandpur ifi
cationofpr ot ei
nswi thsi milarpIv alues.
 Pr oteinChar act erizati
on:I tisempl oy edf ordet ermi ningt hepIofunknown
protei
ns, whi chi sv aluableinfor mat i
onf orchar act eri
zingpr oteins.
 2 D El ectrophor esis:I EF i s of ten used as t he f irstdi mensi on i nt wo-
dimensi onalel ect rophoresis( 2DE)al ongsi de SDS- PAGE as t he second
dimensi on. Thi s al l
ows f or hi ghl y det ailed pr otei n separ ati
on and
characterizat ion.
 Pr otein Fr actionat ion:I EF can be used t of racti
onat e compl ex pr otei
n
mixturesi nt odi stinctpI-basedf ractionsf ordownst ream anal ysis.
 Pr oteinI dent ifi
cat ion:Pr oteinssepar atedbyI EFcanbef urtheranal yzedby
massspect r
omet r
yf orproteini dent i
fication.
 Po st-
transl ationalModi fication Anal ysis:I EF i susef ulf orst udying post -
tr
anslat i
onal modi ficat
ionst hatal terapr otein'
spI .
 Cl i
nicalDi agnost ics:IEFi sappl i
edi ncl i
nicall aborator iesf ort heanal ysisof
serum pr ot ei
ns, hemogl obi nvar i
ant s,andot herbi omar ker s.
 Pr oteinExpr essi onPr ofil
ing:I tisusedt ocompar epr ot einexpr essionl evel
s
andpat ternsi ndi fferentsampl es, suchasdi seasev s.heal t
hyt i
ssues.

6.Two- Dimensi onalGelElectrophoresis(2D-PAGE)

Two- DimensionalGelEl ectrophoresi
s( 2D-PAGE)i sapower fulanal
yti
calt echni
que
usedt osepar ateandanal y
zecompl exproteinmixturesbasedont woi ndependent
properti
es:isoelectri
cpoi nt(pI)andmol ecularweight.Itisastandardmet hodin
proteomics f or st udying pr otein expr ession pat terns, post-
translati
onal
modi fi
cati
ons,andi dent
ifyi
ngpr otei
nswi thinasampl e.Here'
sanov erview of2D-
PAGE:

Pri
nci
ple:
Theprinci
pleof2D- PAGE ist o separateproteinsintwo di
mensions.Thef ir
st
di
mension separ ates pr
otei
ns byt heirisoel
ectri
c poi
nts(pI
),whi
let he second
di
mensionsepar atesthem bythei
rmol ecularweight
s.Thi
stwo-
stepprocessresul
ts
i
nahighlydetai
ledpr otei
nseparat
ion.

KeyComponent s:
1.Isoelectri
cFocusing(IEF)Gel:Inthefir
stdi mension,prot
einsaresepar
atedin
apHgr adi
entgelbasedont heirpIv al
ues.Anel ect
ricfi
eldisappli
edacross
thegel,causingpr
oteinstomi gr
atetot heirrespecti
vepIregionsandbecome
focused.
2.SDS- Polyacrylami
deGel :I
ntheseconddi mensi on,theproteinsfr
om theIEF
gelaref urt
herseparatedinapol y
acry l
ami degelbasedont hei
rmolecul
ar
weights.Thisgeli
stypical
lyunifor
mi npHandcont ai
nstheani oni
cdet
ergent
6
 SDS( Sodium DodecylSulfate)t
odenatureprotei
nsandgivethem anegat
ive
charge,ensur
ingseparati
onbasedonsi zealone.
3.Electr
odes:Two el ectr
odes ar e used t
o apply an el
ectr
icf i
eldi
n both
dimensions.
4.Sampl eLoading:Thepr ot einsampleisappliedtotheI EFgelinthefi
rst
dimension,andthent heIEF-separ
atedprotei
nsareappli
edtot heSDS-
PAGE
gelintheseconddimension.

Pr
ocedure:
1.Isoelectr i
cFocusi ng( FirstDi mensi on) :
o Pr oteinsampl ei sloadedont ot heIEFgel .
o Ane l
ect r
icf ieldi sappl ied,andpr ot ei
nsmi grateaccordingt otheirpI
val ues, becomi ngf ocusedatt hei
rrespect i
vepIposi t
ions.
2.Equi l
ibrat i
on:
o Th eI EFgeli ssoakedi nequi l
ibrati
onbuf f
ertopr epareitfort
hesecond
dimensi on.Thi sbuf fercont ainsreduci ngagent sandSDS.
3.SDS- PAGE( SecondDi mensi on) :
o Th ef ocusedpr oteinsf r
om t heI EFgelar eloadedont ot heSDS- PAGE
gel .
o An e lectri
cf ieldi s appl ied,separ ati
ng proteinsbyt hei
rmol ecular
wei ght s.
4.Det ecti
on:
o Af terel ectrophor esis,thesepar atedpr otei
nscanbev i
sual
izedusi ng
var iousst aining met hods( e.g.,Coomassi e Blueorsi l
verstain)or
transf erredt omembr anesf orWest ernblott
ing.

Applicat i
ons:
 Pr ot einExpr essionPr of il
ing:2D- PAGEi susedt ocompar epr otei
nexpr ession
patter nsi ndi fferentsampl es( e.g.,diseasedv s.healt
hyt i
ssue)andi dentify
dif
f erentiallyexpr essedpr ot eins.
 Pr ot einI dent ifi
cat i
on:Spot scor respondi ng t o separat
ed pr otei
nscan be
exci sed f rom t he gel ,di gest ed wi th enzy mes,and anal y zed using mass
spect romet rytoi denti
fyt hepr ot eins.
 Po st -
Transl ationalModi ficat i
onAnal ysis:2D- PAGEcanr ev ealchangesi n
prot ein i sof orms due t o post -tr
anslational modi f
ications, such as
phosphor y l
ationorgl ycosy lation.
 Pr ot einPur i
fication:Itcanbeusedf orproteinpur if
icat
ion,asspeci f
icpr otein
spot scanbeexci sedf rom t hegel forfurtheranal ysisorfunct ionalstudies.
 Cl i
ni calDi agnost ics:Incl inicall abs,2D- PAGEi susedf oranal yzi
ngser um
prot einsandi dent i
fyi
ngdi seasemar kers.
7.Pulsed- Fi eldGelEl ect r
ophor esi s( PFGE)
Pulsed- Fi
el dGelEl ect r
ophor esis( PFGE)i saspeci ali
zedandpower fultechnique
used f ort he separ ation and anal y si
s ofl arge DNA f r
agment s,such as whol e
chromosomesorl ar gegenomi cf ragment s.Itiscommonl yemploy edi nmol ecular
biol
ogy ,genet ics,andgenomi csf orappl i
cationsl ikeDNA f i
ngerpr int
ing,study i
ng
chromosomalr earrangement s,and epi demi ologicali nvesti
gations. Her e'
s an
overviewofPFGE:

Pri
nci
ple:
PFGEusesanal t
ernat
ingelect
ricf
iel
dthatper
iodi
cal
lychangesdi
rect
ion(pulsed)to
separ
ate large DNA mol ecules based on thei
r size. Unl
i
ke standar d gel
el
ectr
ophoresis,whereDNA f ragmentsmov eli
near
lythroughagelmat r
ix,PFGE
al
lowsforthesepar ati
onofv eryl
argeDNAmol ecul
esbyper i
odi
call
yreversingthe
dir
ect
ionoftheelectri
cfiel
d.
7
KeyComponent s:
1.Agar oseGel :A specialtypeofagar osegelwi t
har elat
ivel
ylow agar ose
concent r
ati
oni susedt oreducer esistanceandf acili
tatethemi grati
onof
l
ar geDNAf ragment s.
2.El ectrodes:Twoset sofelect
rodesar eposi t
ionedonei t
hersideoft hegel.
Theygener atethepulsedelect
ricfield.
3.Pul seControl
ler:Apulsecontroll
err egulatesthedirecti
onanddur at i
onoft he
el
ect ri
cfiel
dpul ses.Ital
lowsf orpr ecisecont r
olov erthemi gr
ationofDNA
fr
agment s.

Pr
ocedur e:
1.Sampl ePr epar ati
on:Genomi cDNA i st ypicallypr epar edf r
om t hesour ce
mat erial(e.g.,
bacter i
alcells)andsubj ectedt or estri
ct i
onenzy medi gestiont o
gener atelargeDNAf r
agment sofint erest.
2.GelLoadi ng:Thedi gestedDNAsampl esar el oadedi ntowel l
satoneendof
theagar osegel .
3.El ectrophor esis:PFGEi schar acterizedbyal ternatingel ectr
icf i
eldpul ses.
Dur i
ngeachpul se,DNAf r
agment sbegi nt omi gr atelinear l
y.Howev er
,bef ore
theycanr eacht heendoft hegel,theel ectri
cf ielddirect i
onr everses,causi ng
theDNA f ragment st ochangedi r ection.Thi spr ocessi sr epeatedmul ti
ple
ti
mes,causi ngtheDNAt omov eina" zi
gzag"f ashiont hr ought hegel.Lar ger
DNA f ragment s mov e mor e slowl yand ar el ess i nfluenced byt he f i
eld
directi
onchanges, al
lowingt hem tosepar at
ef rom smal lerf r
agment s.
4.Det ection:Af terel ectr
ophor esi
s,t he separ at ed DNA f ragment s can be
visualizedusi ngDNAst ainsort r
ansf er r
edt oamembr anef orhy bri
dization
wi t
hspeci f
icprobes, dependi ngont heexper iment algoal s.

Appl
ications:
 DNA F i
ngerprint
ing:PFGE i s used t o create uni que DNA pr ofi
les f or
indi vidual
s orst rains ofmi cr
oorgani
sms,maki ng itv al
uablei nf or ensic
sci enceandepi demi ology.
 Ch r omosomalMappi ng:Itisusedingenet i
csr esearcht omapt heposi ti
ons
ofgenesorspeci ficDNAsequencesonchr omosomes.
 Ge nomi cAnal ysis:PFGEi sempl oyedforst udyinggenomesi ze,det ecti
ng
chr omosomalr ear r
angement s(e.
g.,tr
anslocations),andassessi nggenome
integr it
y.
 E pidemi ologi
calI nvest i
gati
ons:PFGEisusedt ot racet hesourceofi nf
ect i
ous
diseaseout breaksbycompar i
ngtheDNAf ingerprintsofpathogens.
 Cl i
ni calDiagnost ics:Insomecases,PFGEi susedf ordiagnosi nggenet ic
disor derscausedbychr omosomal abnormaliti
es.
 Ge nomeAssembl y:PFGEcanai dint heconst ructi
onofphy sicalmapsf or
assembl i
nglargegenomes.

8.Electr ophor
eticMobi l
it
yShi f
tAssay( EMSAorGelShi f
tAssay )
TheEl ect r
ophoreti
cMobi li
tyShiftAssay( EMSA) ,alsoknownast heGelShi ftAssay
,
i
sawi delyused biochemi caltechniquet o studypr otei
n-DNA i nteract
ions.Itis
empl oyedt oi
nv est
igatethebi ndingofpr otei
ns,typi
callytranscripti
onf actor
s,to
specificDNAsequences.EMSApr ovi
desi nsight
sintothef ormationandst abil
i
tyof
prot
ei n-DNAcompl exesandhel psidenti
fyr egulat
oryelement sinDNAsequences.
Here'sanov er
viewoft heEMSAt echnique:

Pri
nci
ple:
Thepri
ncipl
ebehi
ndEMSAi
sbasedont
hedi
ff
erent
ialmi
grat
ionofDNAmol
ecul
es
8
i
nanel ect
rophor
eticgel,dependingonwhet
hert
heyareboundtoprotei
nsorremai
n
unbound.Whenapr ot
einbindst oaDNAf r
agment,i
talt
erst
heov eral
lchar
geand
shapeoftheDNA- prot
eincompl ex,l
eadi
ngt
ochangesinit
select
rophor
eti
cmobili
ty
comparedtofreeDNA.

KeyComponent s:
1.DNA Pr obe:Thi si s a shor t,r adiolabeled,orf luorescentl
ylabel ed DNA
fragmentcont ai
ningt hespeci ficbi ndingsi t ef ort hepr otei
nofi nterest
.I t
ser v
esasa" bait
"todet ectprotein-DNAi nter
act ions.
2.Pr otei
nExt r
act:Thepr otei
next ractunderst udy ,oftencont aini
ngtranscript
ion
factorsorot herDNA- bi
ndingpr otei ns.
3.GelEl ectrophoresisSy stem:Ty pically,poly acrylami deoragar osegel sare
used.Pol yacryl
ami degelsof ferhi gherr esolutionf orsmal lerDNAf ragment s,
whi l
eagar osegelsaresui tabl
ef orl argerfragment s.

Pr
ocedur e:
1.Pr epar ation ofDNA Pr obe:The DNA pr obe,usual l
ydoubl e-
st r
anded,i s
prepar ed and l abel ed wi t
h a r adioact i
v e i sotope ( e.g.,[ 32P] f or
aut oradiogr
aphy )oraf l
uor escenttag( forfluorescencedet ecti
on).
2.Bi nding React ion:TheDNA pr obei smi xedwi tht hepr ot
einext racti na
bindingbuf fer.Thi sal lowst heproteinst obi ndt ot heirspecifi
cDNAbi nding
sites.
3.El ectrophor esis:Thepr ot ei
n-DNA compl exesandf reeDNA mol ecul esar e
separ atedbyel ectrophor esis.Whenl oadedont oagelandsubj ectedt oan
electricfiel
d,thepr otein-DNAcompl exesmi gratemor esl owlythrought hegel
thanf reeDNAduet ot heirlargersizeandal teredchar ge.
4.Gel Vi suali
zat i
on: Af t
er el ectr
ophor esis, t he gel i s visualized usi ng
aut oradiogr
aphy( forr adioact i
veprobes)oraf luorescenti magingsy stem ( f
or
fl
uor escentlylabel edpr obes) .Ther esulti
ngpat ternr evealswhet herpr otein-
DNAcompl exeswer ef ormed.

Appl
ications:
 I dent if
icat ion ofDNA- ProteinInter actions:EMSA i sused t o confirm the
bindi ngofpr oteins( e.g.,tr
anscr i
ptionf actor s)tospeci ficDNAsequences.
 St udy i
ngDNA- ProteinBi ndingSpeci fi
city:Ithelpsdet erminet hespecif i
cit
yof
apr oteinf ori tsDNAbi ndingsitebyt estingi tsinteract i
onwi thmut atedor
compet i
torDNAsequences.
 Qu anti
f i
cat ion:EMSA can be used t o est i
mat et he bi nding affi
nityand
stoi chiomet ryofpr otein-DNAi nteract i
ons.
 Sc reeni ngf orLi gands:EMSAcanbeempl oy edt oident i
fysmal lmol eculesor
li
gandst hatmodul atepr otei
n-DNAi nteractions.
 Ma ppingPr ot einBi ndingSi t
es:Itassi stsi nmappi ngt hepr eci
sel ocationof
pr oteinbi ndi ngsi tesonDNAsequences.
 Dr ug Di scov er y:EMSA i sused i n drug di scoveryr esearch to screen f or
compoundst hatcandi sruptpr ot
ein- DNAi nt eracti
ons.
 E pigenet ics Resear ch:I tcan be appl ied t o st udy t he effectofDNA
modi fi
cat ions( e.g.,met hylati
on)onpr otein-DNAi nteractions.