30107012 Electrophoresis

8/12/2019 30107012 Electrophoresis

1/25

738 ELECTROPHORESIS Vol. 9

ELECTROPHORESIS

1. Introduction

Electrophoresis is sep r tion techni!ue "ost o#ten pplied to the n l$sis o#%iolo&ic l or other pol$"eric s "ples. It h s #re!uent pplic tion to n l$sis o#proteins '!() nd *+, #r &"ent "i-tures 'see + CLEIC ,CI*S ). The hi&h resolu/tion o#electrophoresis h s " de it 0e$ tool in the d( nce"ent o# %iotechnolo&$ '!().V ri tions o# this "ethodolo&$ re %ein& used #or *+, se!uencin& 'see E+ETICE+ I+EERI+ )2 isol tin& cti(e %iolo&ic l # ctors ssoci ted ith dise ses such sc$stic #i%rosis2 sic0le/cell ne"i 2 "$elo" s2 nd leu0e"i 2 nd est %/lishin&i""unolo&ic l re ctions %et een s "ples on the % sis o# indi(idu l co"/pounds 'see lso C HE4OTHER,PE TICS2 ,+TIC,+CER 5 I44 +O,SS,6 ). Electrophoresis isn e-tre"el$ e##ecti(e n l$tic l tool %ec use it does not necess ril$ ##ect "olecule s structure2 nd it is hi&hl$ sensiti(e to s" ll di##erences in "olecul rch r&e nd " ss.

The ter" electrophoresis re#ers to the "o(e"ent o# solid p rticle throu&h st tion r$ #luid under the in#luence o# n electric #ield. The stud$ o# electro/phoresish s included the "o(e"ent o# l r&e "olecules2 colloids '!()2 #i%ers '!()2 cl $p rticles 'see C L,6S )2 l te- spheres 'see L ,TE TECH+OLO 6 )2 % sic ll$ n$thin& th tc n %e s id to %e distinct #ro" the #luid in hich the su%st nce is suspended. Thiside r n&e in p rticle si e to hich electrophoresis h s %een pplied h s re!uiredth t the theor$ descri%in& electrophoresis %e (er$ &ener l.

The #und "ent l principle %ehind electrophoresis is the e-istence o# ch r&e

sep r tion %et een n$ sur# ce nd the #luid in cont ct ith it. The sur# ce c r/riesn i""o%ili ed ch r&e2 nd the electrol$te #luid in cont ct ith the ch r&ed sur# ce% l nces the electric ch r&e ith n incre sed densit$ o# ions o# the oppo/sitech r&e. ,n pplied electric #ield c n ct on the resultin& ch r&e densities2 c usin&the p rticle to "o(e2 the #luid round the p rticle to "o(e2 or %oth. ,n ppliedelectric #ield lso &ener tes he t2 throu&h resisti(e he tin&2 nd & ses2 throu&helectrol$sis re ctions. E ch is i"port nt in underst ndin& nd desi&n/in& or0in&electrophoresis e!uip"ent.

There re three distinct "odes o# electrophoresis: one electrophoresis2 iso/electric #ocusin&2 nd isot chophoresis. These three "ethods " $ %e used loneor in co"%in tion to sep r te "olecules on %oth n n l$tic l '"L o# "i-turesep r ted) nd prep r ti(e '"L o# "i-ture sep r ted) sc le. Sep r tions in thesethree "odes re % sed on di##erent ph$sic l properties o# the "olecules in the"i-ture2 " 0in& t le st three di##erent n l$ses possi%le on the s "e "i-ture.

*istinction is lso " de "on& electrophoretic techni!ues in ter"s o# the t$peo# " tri- e"plo$ed #or n l$sis. 4 trices include pol$"er &els such s & r/osend pol$ cr$l "ide2 p per2 c pill ries2 nd #lo in& %u##ers. E ch " tri- is used #ordi##erent t$pes o# "i-tures2 nd e ch h s uni!ue d( nt &es.

There re ( riet$ o# techni!ues #or detectin& sep r ted s "ple co"/poundsusin& che"ic l st ins2 photo&r phic "edi 2 nd i""unoche"istr$. E ch detectiontechni!ue lso &i(es di##erent in#or" tion %out the identit$2 !u ntit$2 nd ph$sic lproperties o# the "olecules in the "i-ture. *etection is

Kirk-Othmer Encyclopedia of Chemical Technology. Copyright John Wiley & Sons, nc. !ll rights reser"ed.


2/25


3/25

the


4/25

7@D ELECTROPHORESIS Vol. 9

per"itti(it$ o# the solution:

_ ;

A _

>; _ c ;

_ B; __ oRThere _ A rel ti(e per"itti(it$5 _ o A per"itti(it$ o# #ree sp ce A 8.8 @ _ 1D

_ 1@ CF 'Vc")5 A ( lenc$ o# e ch ion5 c A concentr tion o# e ch ion5 R A 8.31@ GF '"ol )5T A te"per ture2 5 nd > A 9. _ 1D@CF"ol. The rel ti(e per"itti(it$ is "e sure o# the conduct nce o# the pure %ul0 " teri l rel ti(e to ( cuu". In thes lt ter in the %e 0er e- "ple2 the pure %ul0 " teri l ould %e ter2 hich h s rel ti(e per"itti(it$ o# _ 8D.

+ot ll o# the ions in the di##use l $er re necess ril$ "o%ile. So"eti"es thedistinction is " de %et een the loc tion o# the true inter# ce2 n inter"edi teinter# ce c lled the Stern l $er ' ) here there re i""o%ili ed di##use l $er ions2

nd sur# ce o# she r here the %ul0 #luid %e&ins to "o(e #reel$. The potenti l tthe sur# ce o# she r is c lled the et potenti l. The onl$ "ethods ( il %le to"e sure the et potenti l in(ol(e "o(in& the sur# ce rel ti(e to the %ul0. Jec usethe et potenti l is de#ined s the potenti l t the sur# ce here the %ul0 #luid " $"o(e under she r2 this is %$ de#inition the potenti l th t is "e sured %$ thesetechni!ues '3).

The ph$sic l sep r tion o# ch r&e represented llo s e-tern ll$ ppliedelectric #ield #orces to ct on the solution in the di##use l $er. There re t o phe/no"en ssoci ted ith the electric dou%le l $er th t re rele( nt: electrophor/esis2 hen p rticle is "o(ed %$ n electric #ield rel ti(e to the %ul05 ndelectroos"osis2 so"eti"es c lled electroendos"osis2 hen %ul0 #luid "i&r tes ithrespect to n i""o%ili ed ch r&ed sur# ce.

Electro0inetics. The #irst " the" tic l description o# electrophoresis % l ncedthe electric l %od$ #orce on the ch r&e in the di##use l $er ith the (iscous #orces in thedi##use l $er th t or0 & inst "otion ' ). J$ usin& this #orce % l nce2 n e!u tion #orthe (elocit$2 V2 o# p rticle in n electric #ield is

V A _ E B3

here E A the electric #ield stren&th2 VFc"5 nd " A EE o Fh #'0 ) here A et

potenti l2 "V5 h A (iscosit$2 &F'c" s)5 nd A p rticle r dius2 c". The #unction'0 ) is 1 hen the p rticle h s "uch l r&er di "eter th n its dou%le l $er 'hi&hionic stren&ths) nd is 1. #or (er$ lo ionic stren&th solutions here the dou%lel $er is "uch &re ter th n the p rticle di "eter '7). The %eh (ior o# p rticle isco"ple- hen the dou%le l $er nd p rticle di "eter re close to the s "e si e2hich is o#ten the c se '829).

Electroos"otic #lo is lso dependent on the et potenti l t the i""o%i/li ed sur# ce nd the stren&th o# the electric #ield. >or electroos"osis2 the #lo r te&ener ted is

> A __ o

_

_ r ; E B@


5/25

_


6/25

Vol. 9 ELECTROPHORESIS 7@1

here > A #lo r te in "LFs nd r A r dius o# #lo ch nnel in centi"eter.Electroos"otic #lo is &ener ll$ "ini"i ed in pol$"er net or0s such s &els2 %ut isi"port nt in open ch nnels such s c pill ries.

;.;. ener tion o# He t in Electric >ields. One o# the pr ctic l pro%le"sencountered in electrophoresis is the &ener tion o# he t #ro" resisti(e dissip tion o#ener&$ in the electrophoretic "ediu". The &ener tion o# he t '


7/25

the half-cell reactions


8/25

7@; ELECTROPHORESIS Vol. 9

C thode ; H ; O K ; e _ _ ; OH _ K H;

H,

K

OH _ _ , _

K

H; O

_

,node H; O _ ; H K D: O ; K ; e

_

H

K

, _ _ H,

_ Th t is2 ter is electrol$ ed. The h$dro&en & s produced t the c thode c n %eh rdous2 especi ll$ %ec use it is in the (icinit$ o# n electrode th t is lsoproducin& he t. >or this re son2 electrode ch "%ers re usu ll$ open to thet"osphere so th t & ses c n (ent.

The other re ctions t the electrodes produce cid ' node) nd % se 'c th/ode) so th t there is possi%ilit$ o# pH &r dient throu&hout the electrophoresis"ediu" unless the s$ste" is ell %u##ered 'see H 6*RO E+/IO+ ,CTIVIT6 ). Ju##erin&"ust t 0e the current lo d into ccount %ec use the electrol$sis re ctions pro/ceedt the r te o# the current. Electrophoresis s$ste"s so"eti"es "i- nd recir/cul tethe %u##ers #ro" the indi(idu l electrode reser(oirs to e!u li e the pH.

3. 4odes o# Electrophoretic Sep r tions

Mone electrophoresis2 isoelectric #ocusin&2 nd isot chophoresis re ll co""onl$pr cticed s n l$tic l techni!ues. Mone electrophoresis is %$ # r the "ost co"/"onl$ pr cticed nd there re se(er l di##erent one electrophoresis "ethods ndtechni!ues ( il %le #or di##erent sep r tion &o ls. Isoelectric #ocusin& is lso

use#ul #or sep r tion2 nd possi%l$ "ore use#ul #or deter"inin& ch r&ech r cteristics o# s "ple proteins. Isot chophoresis t 0es d( nt &e o# thecontinuit$ o# current cross "ediu" to se&re& te s "ple into conti&uous oneso# hi&h purit$. It is not use#ul s n n l$tic l techni!ue2 %ut is pplied s potenti lprep r ti(e "ethod.

3.1. Electrophoresis E!uip"ent. 4ost electrophoresis e!uip"ent sh res % sic desi&n2 di &r " o# hich is sho n in >i&ure 1. Electrophoresis e!uip"entusu ll$ consists o# t o %u##er reser(oirs2 one #or the node nd one #or the c thode.Ver$ o#ten2 electrophoretic %u##ers h (e % sic pH nd s "ple co"/pounds "i&r te tothe node. The e!uip"ent includes so"e sort o# electrophore/tic "ediu" connectin&the t o reser(oirs2 such s &el2 p per2 or c pill r$ tu%es2 to hich s "ple is pplied.

, direct current po er suppl$ connects the t o electrodes th t re i""ersed in the%u##er reser(oirs. The po er suppl$ is usu ll$ interloc0ed to the electrophoresise!uip"ent throu&h co(er2 isol tin& the "ediu" nd %u##er reser(oirs #ro" hu" ninter ction %ec use cont ct c n %e (er$ d n&erous. S" ll dc currents throu&h thehu" n he rt c n c use c rdi c rrest or rrh$th"i . ,n electric #ield is pplied %et eenthe t o electrode reser/(oirs2 c usin& s "ple co"pounds to "i&r te throu&h the"ediu" nd he t to %e &ener ted in the "ediu". The he t is usu ll$ re"o(ed %$ chilled ter % th or runnin& t p ter. ,t co"pletion o# the electrophoretic run thes "ple ppe rs resol(ed into se(er l di##erent co"ponents lon& the len&th o# the"ediu". In this respect2 &el electrophoresis rese"%les thin/l $er chro" to&r ph$ 'see

C HRO4,TO R,PH6 ).


9/25


Coolin& ter outlet

dc Po er suppl$

C

t h o

d e

% u

# # e r

S "

p l e e

l l s

Electrophoretic " tri-

r e s e r ( o

i r

, n o

d e

% u

# # e r r e s e r ( o

i r

Coolin& %loc0


10/25

7@@ ELECTROPHORESIS Vol. 9

>i&. ;. Mone electrophoresis sep r tion here S2 >2 S 12 nd > 1 re di##erent " teri ls.

co"ponents re co"pletel$ sep r ted into discrete ones s the$ "i&r te throu&hthe "edi onto hich the$ re pplied.

The use o# st nd rds ith s "ples " 0es one electrophoresis p rticul rl$use#ul s n n l$tic l tool. Ho e(er2 hen s "ples c nnot %e n l$ ed on thes "e &el2 di##erences in the e-peri"ent l conditions #ro" e-peri"ent to e-peri/"ent " 0e direct co"p rison "ore di##icult. To " 0e co"p risons #ro" e-peri/"ent to e-peri"ent2 rel ti(e "o%ilit$2 R # 2 is o#ten "e sured %$ "e surin& thedist nce co"ponent tr (els do n the &el co"p red to so"e re#erence or st n/d rd co"ponent.

R# Adist nce "i&r ted %$ co"ponent

B7dist nce "i&r ted %$ re#erence

*isc Electrophoresis. Resolution in one electrophoresis depends criti/c ll$ on&ettin& s "ple co"ponents to "i&r te in #ocused % nd2 thus so"e techni!ues ree"plo$ed to concentr te the s "ple s it "i&r tes throu&h the &el. The "ost co""ontechni!ue is re#erred to s discontinuous pH or disc elec/trophoresis. *iscelectrophoresis e"plo$s t o/&el s$ste"2 here the properties o# the t o &els redi##erent.

*isc electrophoresis s #irst introduced in the e rl$ 19 Ds '11?13) s ( r/ious techni!ues usin& pol$ cr$l "ide &els ere %ein& e-plored nd desi&ned.Ori&in l or0 e"plo$ed se(er l %u##er s$ste"s nd di##erent pol$ cr$l "ide &els inorder to #irst concentr te nd then sep r te co"pounds '1@).

The $ proteins %eh (e in disc electrophoresis s$ste"s depends pri" ril$on di##erences in the pH in the t o &els. The pH o# the %u##ers in %oth the second


11/25

Vol. 9 ELECTROPHORESIS 7@

&el 'the sep r tin& &el) nd the electrode reser(oir re si"il r2 here s the %u#/#erso# the #irst &el 'the st c0in& &el) nd the s "ple itsel# re o# lo er pH. Thisdi##erence in pH llo s #or di##erent s "ple?&el inter ctions. The st c0in& &el isonl$ _ c"2 includin& dist nce #or the s "ple ells2 nd is st c0ed on top o# the

sep r tin& &el hich is _ ;D c" in len&th. ,s ith "ost electrophoretic "ethods2 current is pplied nd the "olecules in the s "ple ells %e&in to "i&r te nod/ ll$.Here2 the "i&r tion is electroche"ic ll$ di##erent %ec use the %u##er in the upperreser(oir ch "%er h s hi&her pH th n th t o# the s "ples nd st c0in& &el. Thisdi##erence in pH llo s the "olecules in the s "ples to "i&r te r pidl$ throu&h thest c0in& &el. =hen the s "ple co"pounds enter the sep r tin& &el2 "o(e"ent isslo ed %ec use o# the pH ch n&e. This #ocuses the "olecules into n rro % ndsnd llo s "ore % nds to %e resol(ed #ro" one nother.

,nother di##erence %et een other t$pes o# electrophoresis nd disc electro/phoresis is th t the "olecules in s "ple do not st rt to si&ni#ic ntl$ sep r te untilenterin& the sep r tin& &el. , discontinuous &el s$ste" " $ %e used ith l"ostn$ t$pe o# one electrophoresis pplic tion.

+ ti(e Mone Electrophoresis. In so"e c ses2 &ood resolution %et eens "ple species c n %e o%t ined ith little or no s "ple pretre t"ent. In these c ses2the &els re s id to %e n ti(e &els. In this "ethod2 the ch r&e on the indi/(idu l s "pleco"ponent is pri" ril$ responsi%le #or its di##erenti l "i&r tion. The rel ti(e "o%ilit$2 R # 2"e sured is proportion l to the ch r&e hen the pore si e in the electrophoretic"ediu" is l r&e co"p red to the co"ponent. =hen the pore si e nd "olecul r si ere %out the s "e2 the si e o# the "olecule %eco"es i"port nt in the electrophoretic"o%ilit$. Jec use o# this "%i&uit$2 the %solute "e nin& o# R # #ro" this techni!ue isuse#ul pri" ril$ #or co"ponent identi#ic /tion nd co"p rison2 nd not #or esti" tin& theproperties o# "olecule.

=hen sep r tin& proteins2 n ti(e one electrophoresis le (es the co"po/nentproteins in #olded2 &lo%ul r st tes2 ith ll su%units int ct. This co"p ct si e " 0es#or # ster runnin& "olecule. + ti(e electrophoresis is use#ul #or resol(in&co"ponents th t " $ di##er %$ s" ll si e ndFor s" ll ch r&e. >or e- "ple2this "ethod s used to isol te &enetic ( ri nts o# so"e proteins in co s "il0'1 )2 hich di##er %$ one ch r&e unit nd ND.1 o# their tot l "ole/cul r ei&ht.This "ode o# electrophoresis c n lso %e used to isol te proteins in n cti(e st te2so th t their cti(ities c n either %e used #or their detection2 or so th t the$ c n %ereco(ered #ro" the &el nd #urther studied.

Reduced S*S Electrophoresis. The co"%in tion o# sodiu" dodec$l 'l ur$l)

sul# te 'S*S) 1 1/;1/3Q2 CH 3 'CH ; )1DCH ; OSO 3+ 2 tre t"ent o# s "ples ndpol$ cr$l "ide &el electrophoresis s #irst descri%ed in the l te 19 Ds '1@21 ). TheS*S is n ionic sur# ct nt th t solu%ili es nd den tures proteins 'see S R>,CT,+TS ).The sur# ct nt co ts protein throu&h h$dropho%ic inter ctions ith the pol$peptide% c0%one2 e##ecti(el$ sep r tin& "ost proteins into their pol$peptide su%units. The"


12/25

7@ ELECTROPHORESIS Vol. 9

those th t re l r&er. The r te t hich "olecules "i&r te throu&h pol$ cr$/l "ide &el is in(ersel$ line r ith the lo& rith" o# their "olecul r ei&ht. Thusden tured s "ples c n %e n l$ ed lon&side st nd rds o# 0no n "olecul rei&ht to id in the interpret tion o# su%st nce s ph$sic l si e.

Other dissoci tin& &ents " $ %e used to #urther %re 0 do n protein. re7/13/ Q " $ %e used to disrupt h$dro&en %onds. =hen ure is the onl$

dissoci tin& &ent dded 'no S*S)2 protein s intrinsic ch r&e is not ##ected ndsep r tion on the % sis o# si e nd ch r&e " $ %e chie(ed. I# protein con/t insintern l disul#ide %onds2 thiol re &ent such s %/"erc ptoeth nol D/;@/;Q " $%e used in order to reduce the s "ple nd %re 0 the disul#ide %onds. Pro/teinsh (in& reduced disul#ide %onds %ind _ 1.@ & o# S*S per &r " o# protein2 co"p redto _ 1 &F& #or nonreduced. T$pic ll$2 %oth reduced nd nonreduced s "ple re runin order to e( lu te protein disul#ide content.

Pulsed >ield el Electrophoresis. The in(erse line r rel tionship %et eenthe rel ti(e electrophoretic "i&r tion o# "olecule nd the lo& rith" o# its si e orlen&th %e&ins to %re 0 do n #or (er$ l r&e "olecul r ei&hts. Pulsed #ield &elelectrophoresis is techni!ue th t s de(eloped to sep r te these l r&e "olecul rei&ht co"pounds2 represented %$ l r&e pieces o# *+, in & rose &els. In pulsed #ieldelectrophoresis2 the direction o# the #ield is inter/"ittentl$ ch n&ed2 either #or rd nd% c0 rd or #ro" side to side. , s" ll "olecule notices no re l di##erence in itselectrophoresis %ec use it c n co"ple/tel$ reorient in e ch #ield direction. Ho e(er2 theredirectionin& o# the electric #ield c uses l r&er "olecules to tr (el in i& & p ttern2puttin& 0in0s into the len&th o# the "olecule. The lon&er the "olecule2 the "ore 0in0s inits len&th2 nd the slo er it tr (els do n the len&th o# the &el. The l r&er "olecule #indsitsel# tr (elin& % c0 rd lon& so"e sections o# its len&th ith respect to the direction

o# the electric #ield. This h s llo ed resolution o# "e& % se si e str nds o# *+,2 ndis one n l$tic l d( nce th t h s " de the Hu" n eno"e Pro


13/25


in one direction or nother2 under the in#luence o# n pplied electric #ield2 until the$re ch one in hich the pH is the s "e s th t "phol$te s isoelectric point. The"phol$te "olecules %u##er the"sel(es nd est %lish the loc l pH s the$ "i&r tethrou&h the &el. ,s the "phol$tes re ch n isoelectric pH2 the$ est %lish

st tion r$ sp ti l pH &r dient in the electrophoretic "ediu".Isoelectric >ocusin&. Isoelectric #ocusin& 'ie#) is n electrophoretic tech/ni!ue

in hich "photeric s "ples re sep r ted ccordin& to their isoelectric points lon& continuous pH &r dient. Ie# n l$ses re c rried out in ( rious " trices: in cr$l "ide2& rose2 nd c pill ries. The & rose or cr$l "ide &els th t re used "ust %e

prep red ith c rrier "phol$tes %r c0etin& speci/#ic pH r n&e. ,#ter so"e ti"e2 the"phol$tes sep r te nd there is pH &r di/ent hich co(ers the r n&e o# ll the"phol$tes isoelectric points. Initi l rese rch on ie# '; ) s pri" ril$ directed to rde( lu tin& the properties o# s$nthetic "phol$tes in solution. L ter or0 re#ined thetechni!ue to ppl$ &el " trices '1;) nd pro(ided the % sis #or ie# "ethodolo&ies.

Pro%le"s ssoci ted ith &el/to/&el ( ri %ilit$ h (e %een recti#ied ith thed( nce"ent o# "phol$te "i-tures. One co""onl$ used "i-ture o# "pho/l$tes2c lled I""o%ilines2 is n i"pro(ed "phol$te "i-ture th t produces no &r dientdri#t or une!u l pH &r dient '; ) nd c n %e used in &el " tri- repro/duci%l$ #ro"one d $ to the ne-t.

Jec use protein s "ples re ctu ll$ "phol$tes2 hen s "ples re lo dedonto the &el nd current is pplied2 the co"pounds "i&r te throu&h the &el untilthe$ co"e to their isoelectric point here the$ re ch ste d$ st te. This techni!ue"e sures n intrinsic ph$sicoche"ic l p r "eter o# the protein2 the pI2 ndthere#ore does not depend on the "ode o# s "ple pplic tion. In ie#2 the hi&hest

s "ple lo d o# n$ electrophoretic techni!ue " $ %e used2 ho e(er2 s "ple lo d##ects the #in l position o# co"ponent % nd i# the lo d is e-tre"el$ hi&h2 ie2 hi&henou&h to titr te the &r dient "phol$tes or distort the loc l electric #ield.

Isoelectric #ocusin& t 0es lon& ti"e '#ro" _ 3 to 3D h) to co"plete %ec uses "ple co"pounds "o(e "ore nd "ore slo l$ s the$ ppro ch the pH in the &elth t corresponds to their isoelectric points. Jec use the &r dient "phol$tes ndthe s "ples stop here the$ h (e no "o%ilit$2 the resisti(it$ o# the s$ste"incre ses dr " tic ll$ to rd the end o# the e-peri"ent2 nd the current decre sesdr " tic ll$. >or this re son2 isoelectric #ocusin& is usu ll$ run ith const nt(olt &e. Const nt current pplic tion c n le d to o(erhe tin& o# the s$ste".

So"e #or"s o# & rose re speci#ic ll$ desi&ned to or0 ith l r&e '"ol tDD2DDD) "olecules ';72;8). The t$pes o# s "ples #or hich the & rose ie#

s$ste" re utili ed re l r&er pl s" proteins such s i""uno&lo%ulins2 or ctu ltissues2 such s or& n s "ples nd tu"ors.

,nother #or" o# ie# is "ethod c lled direct tissue isoelectric #ocusin& 'dti#)';9) here isoelectric #ocusin& in & rose is used to e( lu te tissues. The tissue to%e n l$ ed is pl ced directl$ onto the &el. sin& the tissue itsel# nd not tissuee-tr cts h s d( nced the stud$ o# proteins th t re di##icult to e-tr ct #ro" tissue2or re d " &ed %$ the e-tr ction procedure. *ti# is n i"port nt d( nce"ent in

the re o# s "ple h ndlin& nd pplic tion here direct ppli/c tion o# solid to &el " tri- " $ ctu ll$ enh nce resolution.


14/25

7@8 ELECTROPHORESIS Vol. 9

3.@. Isot chophoresis. Isot chophoresis t 0es d( nt &e o# the # ct th telectroneutr lit$ "ust %e " int ined in n electrophoretic s$ste" in order to support nelectric #ield. I# current p sses throu&h "ediu"2 th t cur/rent "ust %e const nt #ro"one electrode to the other2 re& rdless o# the loc l ion concentr tion or "o%ilit$5 ie2 dilute

ions "ust "o(e # ster to 0eep up ith one o# "ore concentr ted ions. Electric #ieldsco"pens te #or this %ec use the electric #ield stren&th does not h (e to %e const ntlon& the len&th o# the "ediu". The electric #ield stren&th is lo est here the ions re"ost concentr ted nd "ost "o%ile. Isot chophoresis t 0es d( nt &e o# thispheno"enon %$ linin& up the ions o# interest2 # stest '"ost "o%ile) to slo est. This is hi&hl$ speci li ed techni!ue th t re!uires det iled 0no led&e o# the properties o# thes "ple to %e sep r ted2 nd is &ener ll$ not pplic %le to n l$tic l sep r tions o#un0no n constituents.

,n electrophoretic "ediu"2 such s &el or c pill r$ tu%e2 is c st or #illedith n electrol$te th t h s hi&her "o%ilit$ th n n$ co"ponents in the "i-/ture o#interest. This electrol$te2 c lled the le din& electrol$te2 lso #ills the node %u##erreser(oir. , s "ple is pplied on top o# the le din& electrol$te2 nd notherelectrol$te ith lo er "o%ilit$ th n the s "ple #ills the c thode reser/(oir. Thisessenti ll$ en(elopes the s "ple. ,s the electric #ield is pplied2 the le din&electrol$te "o(es r pidl$ to rd the node. The hi&hest "o%ilit$ co"po/nent in thes "ple is dr n to rd the le d electrol$te to #ill in the conducti(it$ & p le#t %$ the!uic0l$ "i&r tin& le d electrol$te. The electric #ield dri(in& the #irst ion in the s "ple"ust incre se to llo the s "ple ion to #ollo t the s "e speed s the le din&electrol$te. The current throu&h the hole "edi is const nt2 so the electric #ieldstren&th incre ses %ec use the resisti(it$ incre ses. The resisti(it$ incre ses

%ec use the #irst ion in the s "ple to #ollo the le d electrol$te dilutes so theelectric #ield ithin the #irst ion s "ple one is incre sed to &i(e the t o ones thes "e speed. Pro&ressi(el$2 e ch ion in the s "ple #ollo s t lo er conducti(it$2hi&her electric #ield2 const nt current densit$2 in ll$2 the tr ilin& electrol$te2 o# lo est"o%ilit$2 ter"in tes the sep r tion.

This sep r tion techni!ue h s %een e"plo$ed pri" ril$ #or prep r ti(e t$peso# sep r tions %ec use det iled 0no led&e o# the properties o# the s "ple isre!uired. ,lso2 %ec use this sep r tion results in discrete ones o# s "ple ions th tre (irtu ll$ pure2 it " 0es sense to use this techni!ue hen the s "ple si e isl r&e. This techni!ue is ine##ecti(e hen the le(els o# i"purities re s" ll ithrespect to the t r&et co"pound5 s" ll "ounts o# s "ple ions do not #or" onesell nd tend to "i- ith the t r&et co"pound. In#or" tion on this techni!ue is( il %le '3D).

@. Electrophoretic 4 teri ls nd 4 trices

V rious support "edi " $ %e e"plo$ed in electrophoretic techni!ues. Sep r /tionon & rose2 cr$l "ide2 nd p per is in#luenced not onl$ %$ electrophoretic"o%ilit$2 %ut lso %$ sie(in& o# the s "ples throu&h the pol$"er "esh. The #inerthe e (e o# selected " tri-2 the slo er "olecule tr (els. There#ore2 "olecul r


15/25


ei&ht or "olecul r len&th2 s ell s ch r&e2 c n in#luence the r te o# "i&r tion.

In ddition to pol$"eric support "edi 2 c pill ries nd #lo in& %u##ers h (e%een used s support "edi #or electrophoresis. ,lthou&h these re not used s

#re!uentl$2 there re de#inite d( nt &es #or cert in t$pes o# s "ples ndpplic tions.

@.1. ,& rose Electrophoresis. ,& rose is produced #ro" the proces/sin& o#red se eed. =hen & r is e-tr cted #ro" the se eed it is in t o co"/ponents2& ropectin nd & rose. The & rose portion is ne rl$ unch r&ed nd is there#ore theportion desir %le #or use s n electrophoretic " tri-. Ch r&e on the & rose le ds toelectroos"otic #lo throu&h the &el. The che"ic l co"posi/tion o# & rose is ltern ti(el$ repe ted residues o# 123/lin0ed/%/ * /& l ctop$r /nosend 12@/lin0ed/32 / nh$dro/ / L/& l ctop$r nose '31).

To prep re &el #or electrophoresis co"%in tion o# & rose nd %u##er ishe ted until the & rose solid is dissol(ed nd %oilin&. The solution is cooled su#/#icientl$ nd then poured into r"ed &el c stin& pp r tus hich #or"s thesh pe o# the &el s it cools. ,#ter the cooled solution is poured into the c stin&pp r tus2 it is llo ed to &el nd c n then %e used in n & rose electrophoresis"ethod. Jec use the co"position o# & rose is net or0 o# residues th t holdter "olecules2 the e-tr & rose solution " $ %e stored nd used t l ter ti"e.

The concentr tion o# & rose "i-tures t$pic ll$ ( ries %et een D. nd ; in ei&htF(olu"e r tio2 lthou&h "ore e-tre"e concentr tions h (e %een e( lu ted.The ( r$in& concentr tions depend on the desired pplic tion. The hi&her theconcentr tion o# & rose2 the s" ller the pore si e.

The use o# & rose s n electrophoretic "ethod is idespre d '3;?3 ). ,n

e- "ple o# its use is in the e( lu tion nd t$pin& o# *+, %oth in #orensics 'see>ORE+SIC CHE4ISTR6 ) nd to stud$ herit %le dise ses '3 ). ,& rose electrophoresis isco"%ined ith other n l$tic l tools such s Southern %lottin&2 pol$"er se ch inre ction2 nd #luorescence. The d( nt &es o# & rose electrophoresis re th t itre!uires no dditi(es or cross/lin0ers #or pol$"eri tion2 it is not h rdous2 loconcentr tion &els re rel ti(el$ sturd$2 it is ine-pensi(e2 nd it c n %e co"%inedith " n$ other n l$tic l "ethods. Pre" de & rose &els re no co""erci ll$( il %le nd !uite con(enient.

@.;. Pol$ cr$l "ide Electrophoresis. Pol$ cr$l "ide &els re s$nthe/ si ed throu&h the co"%in tion o# cr$l "ide 79/ D/1Q '!()2 CH ; CHCO+H ; 2"ono"er nd cross/lin0in& co"ono"er 'see , CR6L,4I*E POL64ERS ). T$pic ll$2 thecross/lin0in& co"ono"er o# choice is +2+ D/"eth$lene%is cr$l "ide 11D/; /9Q'%is cr$l "ide )2 'CH ; CHCO+H) ; 2 lthou&h others re ( il %le2 such s eth$le/nedi cr$l te 'E*,) nd +2+ D/di ll$lt rt rdi "ide '*,T*) 8@77/8 /3Q '37). Thecross/lin0in& o# pol$"eri ed "ono"er ith the co"ono"er is h t controls thepore si e o# the &el pol$"er "esh. This le(el o# pore si e control " 0espol$ cr$l "ide &el electrophoresis n e##ecti(e n l$tic l tool.

The "ost co""onl$ used co"%in tion o# che"ic ls to produce pol$/cr$l "ide &el is cr$l "ide2 %is cr$l "ide2 %u##er2 ""oniu" persul# te2 ndtetr "eth$lenedi "ine 'TE4E*). Joth TE4E* nd ""oniu" persul# te rec t l$sts to the pol$"eri tion re ction. The TE4E* c uses the persul# te toproduce #ree r dic ls2 c usin& pol$"eri tion. Jec use this is #ree/r dic l dri(en

re ction2 the "i-ture o# re &ents "ust %e de& ssed %e#ore it is used.


16/25

7 D ELECTROPHORESIS Vol. 9

The "i-ture pol$"eri es !uic0l$ #ter TE4E* ddition2 so it should %e poured intothe &el/c stin& pp r tus s !uic0l$ s possi%le. Once the &el is poured into prep red #or"2 co"% c n %e pplied to the top portion o# the &el %e#ore pol$/"eri tion occurs. This co"% sets s" ll indent tions per" nentl$ into the top

portion o# the &el hich c n %e used to lo d s "ples. I# the co"% is used2 s "plesre then t$pic ll$ "i-ed ith he (ier solution2 such s &l$cerol2 %e#ore the s "pleis pplied to the &el2 to pre(ent the s "ple #ro" dispersin& into the reser/(oir%u##er.

4 -i"u" resolution o# proteins is the " in consider tion hen deter"in/in&the cr$l "ide concentr tion o# &el. T o p r "eters de#ine the co"position o#&el: the t o# tot l "ono"er2 T ' cr$l "ide plus %is cr$l "ide in &r "s per1DD "L)2 nd the proportion %$ ei&ht o# "ono"er th t is the cross/lin0in& &ent2 C '%is cr$l "ide) '38). els h (in& concentr tions %et een nd 1 ret$pic ll$ used to chie(e the "ost desir %le sep r tion o# the co"ponents o#interest. nli0e the & rose " tri-2 once the "i-ture pol$"eri es2 it c nnot %ereused. These &els re popul r %ec use o# their dur %ilit$. The$ c n %e dried nds (ed2 lthou&h the$ re t$pic ll$ photo&r phed or sc nned electronic ll$ #orrchi( l purposes these d $s. Prepoured pol$ cr$l "ide &els o# ( rious T ndC re lso co""erci ll$ ( il %le.

Pol$ cr$l "ide &el electrophoresis is one o# the "ost co""onl$ used elec/trophoretic "ethods. ,n l$tic l uses o# this techni!ue center round proteinch r cteri tion2 e&2 purit$2 si e2 or "olecul r ei&ht2 nd co"position o# pro/tein. Pol$ cr$l "ide &els c n %e used in %oth reduced nd nonreduced s$ste"s sell s in co"%in tion ith discontinuous nd ie# s$ste"s '39).

,n e- "ple o# the use o# pol$ cr$l "ide &els in n ie# s$ste" in co"%in /tion

ith i""uno%lottin& is &i(en in '@D)2 here this "ethod is used to detect lo!u ntities o# &roup speci#ic co"ponent ' C) su%t$pes. ,nother e- "ple o# pol$/cr$l "ide in co"%in tion ith ie# is &i(en in '@1)2 here the techni!ue is used s screenin& tool #or inherit nce o# cert in pol$"orphic protein.

Joth the e se o# use o# this "ethod #or ch r cteri tion o# proteins ndnucleic cids2 nd the %ilit$ to n l$ e " n$ s "ples si"ult neousl$ #or co"/p r ti(e purposes2 h (e led to the pre( lence o# this techni!ue. The dr % c0s o# pol$ cr$l "ide " tri- is th t cr$l "ide is neuroto-in2 the re &ents "ust %eco"%ined e-tre"el$ c re#ull$2 nd the &els re not s pli %le s "ost & rose &els.

@.3. P per Electrophoresis. P per '!() s n electrophoretic " tri- se"plo$ed in so"e o# the #irst electrophoretic techni!ues de(eloped to sep /r teco"pounds. P per is e sier th n &el " tri- %ec use the p per " tri- re!uires noprep r tion. Jesides %ein& e s$ to o%t in2 p per is &ood "ediu" %ec use it does notcont in " n$ o# the ch r&es th t inter#ere ith the sep r /tion o# di##erent co"pounds.T o t$pes o# p per e"plo$ed in this t$pe o# electro/phoresis re =h t" n 3 44 'D.3"") nd =h t" n +o. 1 'D.17 "").

In p per electrophoresis2 the s "ple is pl ced directl$ onto chro" to/&r phicor #ilter p per nd then e-posed to %u##er solution t e ch end nd n electric#ield is pplied. ,s in "ost electrophoretic techni!ues2 ch r&ed d$es re co"%inedith s "ples nd st nd rds to see the pro&ress o# the electrophor/esis. The

"o(e"ent o# s "ples on p per is %est hen the current #lo is p r llel to the #i%er-is in the p per. The p per h s hi&h resist nce so (olt &es re


17/25

Vol. 9 ELECTROPHORESIS 7 1

t$pic ll$ "uch hi&her th n in & rose nd pol$ cr$l "ide " trices. Li0e & r/osend pol$ cr$l "ide " trices2 p per is co"%ined ith other n l$tic l tools toenh nce sep r tion nd identi#ic tion o# s "ple co"ponents. >or e- "ple2 p perelectrophoresis h s %een co"%ined ith chro" to&r ph$ '@;).

The di##erence %et een p per electrophoresis nd p per chro" to&r ph$ isth t electrophoresis sep r tes %$ ch r&e2 here s chro" to&r ph$ sep r tes %$pol rit$. This co"%ined techni!ue s used to e( lu te pol$"orphis"s o# thehe"o&lo%in "olecule2 ie2 nor" l ,/t$pe (ersus the sic0le cell S/t$pe. It h s %eenc lled peptide #in&erprintin&. This "ethod s l ter "odi#ied '@3) to #urthere( lu te peptides nd is one techni!ue 0no n s peptide " ppin&. The peptide" ppin& techni!ue lso uses hi&h (olt &es to o%t in the desired resolution.

So"e d( nt &es o# p per in electrophoresis re th t p per is re dil$ ( il/%le2 e s$ to h ndle2 nd ne "ethodolo&ies c n %e de(eloped r pidl$. The dis/d( nt &es o# p per electrophoresis re th t the porosit$ o# p per c nnot %econtrolled2 the techni!ue is not (er$ sensiti(e2 nd it is not e sil$ reproduci%le.

@.@. C pill r$ Electrophoresis. C pill ries ere #irst pplied s sup/port"ediu" #or electrophoresis in the e rl$ 198Ds '@@2@ ). The &l ss c pill ries used ret$pic ll$ ;D?;DD "" in di "eter '@ )2 " $ %e #illed ith %u##er or &el2 nd re #re!uentl$co ted on the inside. C pill ries re used %ec use o# the hi&h sur# ceF(olu"e r tio th tllo s r pid he t dissip tion nd thus hi&her (olt &es th n tr dition l "ethods. Theonl$ li"it tions ssoci ted ith c pill ries re li"its o# detection nd cle r nce o#s "ple co"ponents.

Li"its o# detection %eco"e pro%le" in c pill r$ electrophoresis %ec use the"ounts o# n l$te th t c n %e lo ded into c pill r$ re e-tre"el$ s" ll. In ;D/"" c pill r$2 e&2 there is D.D3/"LFc" c pill r$ len&th. This is 1F1DD? 1F1DDD o# the

(olu"e t$pic ll$ lo ded onto pol$ cr$l "ide or & rose &els. >or tr ce n l$sis2 (er$ s" ll nu"%er o# n l$te "olecules " $ ctu ll$ e-ist in the c pill r$ #ters "ple lo din&. To detect these s" ll "ounts o# n l$te2 so"e on/line detectorsh (e %een de(eloped hich use conducti(it$2 l ser *oppler e##ects2 or n rro l$#ocused l sers '!() to detect either %sor% nce or #luorescence '@72@8). Theconducti(it$ detector cl i"s detection li"its do n to 1D "olecules. The l ser%sor% nce detector h s %een used to "e sure so"e o# the constituents in sin&le hu" n cell 'see T R,CE ,+* RESI* E ,+,L6SIS ).

Cle r nce o# s "ple co"ponents #ro" c pill r$ is pro%le" th t h s not%een s ell resol(ed s detection. su ll$2 c pill r$ electrophoresis is conductedin &l ss c pill r$ th t is co ted on its inner sur# ce. So"e pplic tions c ll #or

&el/#illed c pill ries. C pill ries re di##icult to co t or #ill ith &el2 nd re tooe-pensi(e to disc rd #ter e ch use2 s is done hen usin& st nd rd &el. There/#ore2 ll the s "ple h s to %e cle red #ro" the c pill r$ %e#ore the c pill r$ is used#or nother s "ple2 "uch li0e n l$tic l chro" to&r ph$. This c n %e pro%le"hen c pill ries re so"eti"es "ore th n 1DD c" lon&. ,nother pro/%le" riseshen n l$tes %sor% to the &l ss 'or co ted) lls in the c pill r$2 #re!uentoccurrence. This l tter pro%le" h s pre(ented c pill r$ electrophor/esis #ro"%eco"in& pr ctic l "ethod #or protein n l$sis. In co ted c pill ries2electroos"osis lon& the c pill r$ lls so"eti"es dds (elocit$ to the n l$tes2nd cle r nce is less o# n issue. , &re t de l o# rese rch h s #ocused on

choosin& nd opti"i in& c pill r$ co tin&s th t resist protein #oulin& nd %sor%su##icient ch r&e #ro" %u##er ions to &i(e electroos"otic #lo .


18/25

7 ; ELECTROPHORESIS Vol. 9

Electroos"otic #lo in c pill r$ lso " 0es it possi%le to n l$ e %othc tions nd nions in the s "e s "ple. The onl$ re!uire"ent is th t the electro/os"otic #lo do nstre " is o# &re ter " &nitude th n electrophoresis o# theoppositel$ ch r&ed ions upstre ". Electroos"osis is the pre#erred "ethod o# &en/

er tin& #lo in the c pill r$2 %ec use the ( ri tion in the #lo pro#ile occurs ithin #r ction o# "icron #ro" the ll '@9). =hen electroos"osis is used #or s "plelo din&2 di##erin& "ounts o# n l$te c n %e #ound %et een the s "/ple in thec pill r$ nd the unin


19/25


co"%inin& t o di##erent electrophoresis "ethods in t o/di"ension l ';*) elec/trophoretic techni!ues.

.1. Che"ic l St inin&. St inin& techni!ues th t help to (isu li e % ndin&p tterns resultin& #ro" electrophoresis ( r$ ' @? ). The si e nd t$pe o# the

co"pound s ell s the electrophoretic " tri- dict te nd o#ten li"it the ( riet$ o#st ins th t c n %e used. 4olecules c n %e lost durin& the st inin& process2 so "ostst inin& procedures incorpor te #i-in& step either %e#ore or in con


20/25

%se ith electro-phoresis in $oth the clinical and research settings.


21/25

7E@ ELECTROPHORESIS Vol. 9

,% ,& ,&?,% precipitin

,&?,% precipitin

,& ,&

(a) (b)

,&?,% precipitin ,%/cont inin& &el ,&?,% precipitin

1 : ;

1 : @

1 : 8

1 : 1

I

1 : 3

;

S " p

l e

St nd rds >irst &el S "ple ori&in

(c) (d)

>i&. 3. I""unolo&ic l re ctions2 here ,& is nti&en nd ,% is nti%od$2 #or detection inelectrophoresis: ' ) Ouchterlon$ techni!ue5 '%) sin&le/r di l di##usion5 'c) roc0et i""u/noelectrophoresis5 nd 'd) crossed i""unoelectrophoresis.

The dou%le/i""unodi##usion techni!ue2 o#ten re#erred to s the Ouchterlon$techni!ue2 uses n & rose &el s the " tri-. Holes re " de in the & rosehere either s "ple or ntiser is pl ced. The t o solutions re llo ed to di#/#useinto the " tri- #or predeter"ined ti"e. I# there is re ction %et een the nti&enin the s "ple nd the nti%od$2 precipit te is #or"ed. The ,& _ ,% re c/tion linec n %e (isu li ed #ter st in is used '>i&. 3 ). Si"il rl$2 the sin&le/r di li""unodi##usion '>i&. 3%) techni!ue is re ction o# e!uili%riu" %et een nti&ennd nti%od$. The di##erence is th t the nti&en is dded to r"ed & rosesolution %e#ore it &els. Holes re cut in the &el s #or the Ouchterlon$ techni!uehere s "ples re pl ced. The s "ple di##uses into the &el nd2 i# re cti(e ith thenti%od$2 #or"s n re o# precipit tion round the hole th t is (isu li ed #terst inin&.

The 4 ncini nd Ouchterlon$ techni!ues re the % sis o# the techni!uese"plo$ed in i""unoelectrophoresis. , techni!ue re#erred to s roc0ets ' 9) isn "ed s such %ec use o# the ppe r nce o# roc0et/sh ped nti&en? nti%od$

precipitin #or"ed #ter n nti&enic s "ple is electrophoresed throu&h &el/cont inin& nti%od$ '>i&. 3c).


22/25

Vol. 9 ELECTROPHORESIS 7

,nother #re!uentl$ used "ethod o# i""unoelectrophoresis is techni!ue0no n s crossed/i""unoelectrophoresis '>i&. 3d) ' 9). , s "ple is #irst run (er/tic ll$ throu&h n & rose &el #or predeter"ined ti"e. Secondl$2 the &el rehere the s "ple s electrophoresed is t$pic ll$ cut out nd pl ced hori on/t ll$

into si"il rl$ si ed re o# n nti%od$/cont inin& &el. ,s n electric l current ispplied to the second &el s$ste"2 the s "ple in !uestion electrophor/eses throu&hthe &el nd #or"s n nti&en? nti%od$ precipitin o(er n re th t ( ries #ro" s" llto l r&e2 dependin& on the % ndin& p ttern o# electrophoresis #ro" the #irst &els$ste".

,t so"e h t "ore % sic le(el2 %oth & rose nd cr$l "ide &el s$ste"sh (e %een used #or direct i""uno#i- tion. In these &els2 s "ples re electrophor/esed nd then i""uno#i-ed %$ either usin& strips o# cellulose cet te so 0ed in nnti%od$ or the nti%od$ is pl ced directl$ o(er the s "ple re o# the &el.

,ll o# these techni!ues re "ost o#ten2 %ut not e-clusi(el$2 used in the clin/ic lsettin& in order to di &nose %nor" lities or to e( lu te inherit nce p tterns o#pol$"orphic proteins. 4 n$ pplic tions o# these techni!ues e-ist ' D? @).

.3. T o/*i"ension l Electrophoresis. T o/di"ension l ';*) elec/trophoresis is uni!ue2 o##erin& n n l$tic l "ethod th t is %oth reproduci%le ndsensiti(e. It is re#erred to s ;* %ec use it e"plo$s t o di##erent "ethods o#electrophoresis2 in t o di##erent di"ensions2 to produce one result. E ch "ethodsep r tes the s "ple co"pounds % sed on di##erent properties o# e ch co"pound. Theco"%in tion o# the t o "ethods &i(es %etter resolution o# the co"pounds in the s "pleth n could %e chie(ed ith either "ethod lone. >or e- "ple2 e ch "ethod lone " $sep r te up to 1DD co"ponents o# s "ple2 here s to&ether the$ " $ sep r te up to1D2DDD co"ponents.

, p ir o# electrophoretic techni!ues co""onl$ e"plo$ed in ;* n l$ses reisoelectric #ocusin& 'ie#) nd S*S?pol$ cr$l "ide &el electrophoresis 'S*S? p &e).Ie# sep r tes s "ple co"pounds ccordin& to isoelectric point2 here s S*S?p &e sep r tes the co"pounds %$ "olecul r ei&ht. , ;* n l$tic l tech/ni!ueusin& ie# nd S*S?p &e to sep r te tot l protein results in &el h (in& % nds orspots in r ndo" p ttern ' ). E ch spot represents uni!ue co"ponent o# s "ple. , sin&le ch r&e di##erence in co"ponent c n %e identi/#ied on the &el %$ uni!ue spot. This propert$ o# ;* electrophoresis2 hich llo s identi#ic tion o#identic l proteins th t di##er %$ one ch r&e di##erence2 h s " de it n in( lu %letechni!ue #or the "olecul r &enetic co""unit$. So#t re is ( il %le to identi#$e ch spot on &el nd co"p re %nor" lities in s "ples such s hu" n %lood

' )2 Escherichi coli2 nd $e st proteins..@. Jlottin& Techni!ues. Pro%le"s encountered hen tr$in& to n /

l$ e resol(ed co"ponents o# s "ple "i-ture on &el2 ith techni!ues such sdirect i""uno#i- tion or pplic tion o# li& nd2 c n %e circu"(ented ith the useo# %lottin& techni!ues #ollo ed %$ st inin& or utor dio&r ph$. It s the in %ilit$ o#so"e co"pounds2 such s nti%odies or li& nds2 to enter the &el " tri- o# speci#ic &el s$ste" th t led to the de(elop"ent o# ( rious %lottin& techni!ues hichh (e %eco"e idespre d since the l te 197Ds. The nucleic cid nd protein%lottin& techni!ues h (e %eco"e use#ul %ec use these co"%ine electrophoreticn l$ses nd sensiti(e i""unolo&ic l tools. , %lottin& techni!ue in(ol(es thetr ns#er o# nucleic cids or proteins2 i""edi tel$ #ter %ein& sep /r ted %$electrophoresis2 #ro" the &el " tri- to nother " tri-. T$pic ll$2 the


23/25

7 ELECTROPHORESIS Vol. 9

other " tri- is nitrocellulose p per2 n$lon2 or other hi&h ##init$ "e"%r ne nd the"ode o# tr ns#er is electrotr ns#er #or proteins nd c pill r$ tr ns#er #or nucleiccids. Once nucleic cids or proteins re tr ns#erred2 #urther n l$ses c n %eper#or"ed. On nitrocellulose p per or n$lon2 nucleic cids nd proteins re "ore

ccessi%le th n in the ori&in l &el " tri-. This second " tri- is then tre ted ith li& nd to identi#$ speci#ic co"ponent o# s "ple.

>or e- "ple2 the techni!ue o# Southern %lottin& s de(eloped ' 7) #or useith & rose &el electrophoresis o# *+, #r &"ents. Southern %lots re desi&ned todetect speci#ic se!uences o# *+,. ,#ter electrophoresis is co"plete2 the *+, isden tured nd the sin&le/str nded *+, tr ns#erred to the speci ll$ prep rednitrocellulose p per. The nitrocellulose is then incu% ted ith r dio cti(e R+, or*+, co"ple"ent r$ to those *+, se!uences o# interest. ,#ter the nitrocellu/loseh s %een su##icientl$ incu% ted ith the r dio cti(e co"ple"ent r$ *+,2utor dio&r ph$ is used to identi#$ the #r &"ents o# interest.

The +orthern %lottin& techni!ue is si"il r to the Southern %lottin& techni/!ueith the e-ception th t +orthern %lots detect speci#ic se!uences o# R+,2 not *+,.

The techni!ue o# i""uno%lottin&2 o#ten re#erred to s =estern %lottin&2 is#re!uentl$ used #or ( riet$ o# pplic tions here protein concentr tions re lond st inin& o# the electrophoretic " tri- does not produce de!u te resolution. Inthese inst nces2 s ith Southern %lottin&2 proteins re tr ns#erred to sec/ond" tri- li0e nitrocellulose or n$lon nd re then tre ted ith ntiser speci#ic to desired protein ' 82 9).

Jlottin& techni!ues " $ %e used in ( riet$ nd co"%in tion o# electro/phoretic s$ste"s th t " 0es their use idespre d nd con(enient hen protein

concentr tions re "ini" l nd & rose or pol$ cr$l "ide is the " tri- choice.

JIJLIO R,PH6

Electrosep r tions2 Electrophoresis 2 in ECT @th ed.2 Vol. 92 pp. 3 ?37 2 %$ Scott Rud&end thleen 4 r0e$2 S$ner&en2 Inc.5 Electrosep r tions2 Electrophoresis in ECT 'online)2postin& d te: *ece"%er @2 ;DDD2 %$ Scott Rud&e nd thleen 4 r0e$2 S$ner&en2 Inc.

CITE* P JLIC,TIO+S

1. G. S. +e " n2 Electroche"ic l S$ste"s2 Prentice H ll2 Inc.2 En&le ood Cli##s2 +. G.1973.

;. R. ,. Ro%inson nd R. H. Sto0es2 Electrol$te Solutions2 Jutter orths Scienti#icPu%lic tions2 London2 19 9.

3. R. G. Hunter2 in R. H. Otte ill nd R. L. Ro ell2 eds.2 Met Potenti l in Colloid Science2Principles nd ,pplic tions2 ,c de"ic Press2 Inc.2 +e 6or02 1981.

@. S. R. Rud&e nd P. Todd2 in 4. R. L disch2 R. C. =illson2 C. C. P inton2 nd S. E.Juilder2 eds.2 Protein Puri#ic tion2 >ro" 4olecul r 4ech nis"s to L r&e/Sc leProcesses2 ,CS S$"posiu" Series @;72 = shin&ton2 *.C. 199D2 p. ;@@.

. O. Stern2 Meitschr. Ele0troche". 3D2 D8 '19;@).

. 4. (on S"olucho s0i2 Jull. ,0 d. Sci. Cr co(ie2 Cl sse Sci. 4 th. + tur. 12 18; '19D3).


24/25


7. E. Hu c0el2 Ph$si0. Meitschr. ;@2 ;D@ '19;@).8. *. C. Henr$2 Proc. R. Soc. 'London) Ser. , 1332 1D '1931).9. R. =. O Jrien nd L. R. =hite2 G. Che". Soc. > r d $ II 7@2 1 D '1978).

1D. . H nni&2 H. =irth2 nd E. Schoen2 ,pollo/So$u Test Pro. C rle2 4. >r n02 nd 4. V. Olson2 Science ;3;2 '198 ).

;D. . >. C rle nd 4. V. Olson2 +ucleic ,cids Res. 1;'1@)2 @7 '198@).;1. . Chu2 *. Vollr th2 nd R. =. * (is2 Science ;3@2 1 8; '198 ).;;. >. S. Collins nd co/ or0ers2 Science ;3 2 1D@ '198 ).;3. S. . L rence2 C. L. S"ith2 R. Srin st ( 2 C. R. C ntor2 nd S. 4. =eiss" n2 Science

;3 2 1387 '198 ).;@. O. Vester%er&2 ,ct Che". Sc nd. ;32 ; 3 '19 9).; . H. S(ensson2 ,ct Che". Sc nd. 1 2 3; '19 1).; . ,. Chr "% ch2 The Pr ctice o# Uu ntit ti(e el Electrophoresis2 VCH Pu%lishers2

*eer#ield Je ch2 >l .2 198 2 p. 1@1.;7. ,. Rosen2 . E02 nd P. ," n2 G. I""unol. 4ethods ;82 1 '1979).;8. C. ,. S r (is2 4. O Jrien2 nd +. M "chec02 G. I""unol. 4ethods ;92 97 '1979).;9. C. ,. S r (is nd +. M "chec02 G. I""unol. 4ethods ;92 91 '1979).3D. >. 4. E(er erts2 >. E. P. 4i00ers2 nd Th. P. E. 4. Verhe&&en2 Sep. Puri#. 4eth. 2 ;87

'1977).31. C. ,r 0i2 Jull. Che" Soc. Gpn. ;92 @3 '19 ).3;. C. ,. ,lper2 in =. G. =illi "s2 E. Jeutler2 ,. G. Ersle(2 nd 4. ,. Licht" n2 eds.2

He" tolo&$2 3rd ed.2 4c r /Hill Joo0s2 Inc.2 +e 6or02 19832 p. 1 1 .33. E. S. L nder2 + ture 'London) 3392 D1 '1989).3@. T. 4 ni tis2 E. >. >ritsch2 nd G. S "%roo02 4olecul r Clonin&2 Cold Sprin& H r%or

L %or tor$2 Cold Sprin& H r%or2 +e 6or02 19832 pp. 1 D?171.3 . R. >. Jo$er2 4odern E-peri"ent l Jioche"istr$2 The Jen< "inFCu""in&s Pu%lish/in&

Co.2 Inc.2 198 2 p. ;19.3 . S. =ood nd S. L n&lois2 G. Chro" to&r. 92 @;1 '1991).37. Re#. ; 2 p. @.38. *. E. r#in2 4eth. En $"ol. 18;2 @;8 '199D).39. G. >. Ro%$t nd J. G. =hite2 Jioche"ic l Techni!ues: Theor$ nd Pr ctice2 = (el nd

Press2 Inc.2 Prospect Hei&hts2 Ill.2 1987.@D. S. ,. =est ood2 Electrophoresis 2 '198 ).@1. J. Judlo e nd R. S. 4urch2 Electrophoresis 2 ;3 '198 ).@;. V. In&r "2 Jiochi". Jioph$s. ,ct ;82 39 '19 8).@3. ,. t 2 =. *re$er2 nd C. ,n#insen2 G. Jiol. Che". ;3@2 8; '19 9).@@. G. =. Gor&enson nd . *. Lu0 cs2 ,n l. Che". 32 1;98 '1981).@ . G. =. Gor&enson nd . *. Lu0 cs2 Science ;;;2 ; '1983).@ . 4. G. ordon2 . Hu n&2 S. L. Pentone$2 Gr.2 nd R. +. M re2 Science ;@;2 ;;@ '1988).@7. . Hu n&2 T/ . G. P n&2 4. G. ordon2 nd R. +. M re2 ,n l. Che". 92 ;737 '1987).

@8. 6./>. Chen& nd +. G. *o(ichi2 Science ;@;2 ; '1988).


25/25

7 8 ELECTROPHORESIS Vol. 9

@9. V. Pretorius2 J. G. Hop0ins2 nd G. Schie0e2 G. Chro" to&. 992 ;3 '197@).D. T. Tsud 2 . +o"ur 2 nd . + 0 & 2 G. Chro" to&. ; @2 38 '1983).1. 4. Jier2 in G. ,. ,sen. I(or$2 ,IChE. G. 3@2 @7@ '1988).@. I. S$ro($ nd M. Hodn$2 G. Chro" to&. 92 17 '1991).. T. R %illoud2 Electrophoresis 112 78 '199D).. C. R. 4erril2 4. . H r se $ch2 nd 4. . H rrin&ton2 in 4. G. *unn in Re#. 132

p. 3;3.7. O. Ouchterlon$2 ,ct P thol. 4icro%io. Sc nd. ; 2 18 '19@8).8. . 4 ncini2 G. P. V er" n2 ,. O. C r%on r 2 nd G. >. Here" ns2 Protides Jiol. >luids

112 37D '19 3).9. C./J. L urell2 ,n l. Jioche". 1 2 @ '19 ).D. M. L. , deh nd C. ,. ,lper2 Proc. + tl. ,c d. Sci. 77' )2 3 7 '198D).

1. G. P. 4cCue2 R. H. Hein rd2 nd R. Tenold2 Re(. In#ect. *is. 8'S@)2 S37@ '198 ).;. R. G. Micc rdi2 Clinic l L %or tor$ Techni!ues #or the 198D s2 ,l n R. Liss2 Inc.2 +e

6or02 198D2 p. @33.3. C. ,. ,lper nd R. >. Ritchie2 in R. >. Ritchie2 ed.2 ,uto" ted I""uno n l$sis2 4 rcel

*e00er2 Inc.2 +e 6or02 19782 p. 139.@. C. ,. ,lper nd ,. 4. Gohnson2 Vo- S n&. 172 @@ '19 9).. P. H. O > rrell2 G. Jiol. Che". ; D'1D)2 @DD7 '197 ).. G. E. Celis2 J. Honore2 . J u 2 nd G. V nde0erc0ho(e2 JioEss $s 1;';)2 93 '199D).7. E. Southern2 G. 4ol. Jiol. 982 D3 '197 ).8. *. *. *$0es nd H. >. Poles0$2 Electrophoresis 2 ;1 '198 ).9. J. Jountin2 S. H. >en&2 nd P. ,rn ud2 ,". G. Hu". enet. 372 1D98 '198 ).

E+ER,L RE>ERE+CES

R. C. ,llen2 C. ,. S r (is2 nd H. R. 4 urer2 el Electrophoresis nd Isoelectric >ocusin& o#Proteins2 = lter de ru$ter2 +e 6or02 198@.

,. Chr "% ch2 The Pr ctice o# Uu lit ti(e el Electrophoresis2 VCH Pu%lishers2 +e 6or02198 .

J. ,. J ldo nd E. R. To(e$2 Protein Jlottin&: 4ethodolo&$2 Rese rch2 nd *i &nostic ,pplic tions2 r&er2 S it erl nd2 1989.

G. >. Ro%$t nd J. G. =hite2 Jioche"ic l Techni!ues: Theor$ nd Pr ctice2 = (el ndPress2 Inc.2 Prospect Hei&hts2 Ill.2 1987.

4. G. *unn2 ed.2 el Electrophoresis o# Proteins2 =ri&ht2 Jristol2 . .2 198 .+. C tsi"pool s2 4ethods o# Protein Sep r tion2 Plenu" Press2 +e 6or02 197 .R. >. Jo$er2 4odern E-peri"ent l Jioche"istr$2 The Jen< "inFCu""in&s Pu%lishin& Co.2

Inc.2 198 .R. G. Hunter2 in R. H. Otte ill nd R. L. Ro ell2 eds.2 Met Potenti l in Colloid Science2

Principles nd ,pplic tions2 ,c de"ic Press2 Inc.2 +e 6or02 1981.

S COTT R * E>eR- Incorpor ted

30107012 Electrophoresis

Documents