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7/21/2019 Western Blot Analysis
http://slidepdf.com/reader/full/western-blot-analysis-56d81a67053e2 1/7
Separation and analysis of proteins extracted from Torenia sp.,
Saccharomyces cerevisiae, Gallus gallus domesticus and Salmo
salar using gel-electrophoresis and Western blot
Goh Xin Rong and Lim Yi FeiM15606 11 August 2015
BL6404 Advanced Biochemsitr
INTRODUTION
!he se"aration o# "roteins through "o$acr$amide ge$ e$ectro"horesis %&AG'( is one o# the
#undamenta$s o# "roteomics) !his mo$ecu$ar sieving ana$tica$ techni*ue sets the stage #or various
research a""$ications such as the stud o# "rotein e+"ression $eve$s as ,e$$ as c$inica$ a""$ications in
the identi#ication o# "rognostic -iomar.ers and disease di##erentiators /1) !he -asis #or &AG' is that a
charged mo$ecu$e ,ou$d migrate to,ards a termina$ o# o""osite "o$arit in an a""$ied e$ectric #ie$d) n "roteomics stud the sam"$es are o#ten denatured and treated ,ith sodium dodec$ su$#ate %33()
!his ensures that the "rotein sam"$es ,i$$ -e uni#orm$ charged) !hese ge$s are o#ten termed as non
native &AG')
!"ica$$ most "rotoco$s s"eci# the need o# a stac.ing and reso$ving ge$ #or 33&AG') n$i.e the
reso$ving ge$ the stac.ing ge$ has a $arger "ore si7e made "ossi-$e - its $o,er acr$amide content
and $o,er "8 t"ica$$ o# "8 6)9) t a$so "ossesses a di##erent ionic content) :hi$e the reso$ving ge$
is res"onsi-$e #or se"arating the "roteins into their res"ective mo$ecu$ar ,eights the stac.ing ge$
ensure that a$$ the "roteins ,i$$ simu$taneous$ run in a common -and) !his stac.ing e##ect is achieved
through the inter"$a o# t,o ma;or ions ch$oride and g$cine /2)<,ing to their high masstocharge
ratio ch$oride ions "resent in the stac.ing ge$ are high$ mo-i$e and ,ou$d -e the #irst to migrateto,ards the anode) <n the other hand g$cine ,hich is "resent in the running -u##er ,ou$d -e the $ast
to migrate to,ards the anode) As a ,ea. acid g$cine can e+ist either as an uncharged 7,itterion at
$o, "8 or negative$ charged g$cinate anion at higher "8 /=) uring 33&AG' g$cine migrates
a,a #rom the cathode as the move to,ards the stac.ing ge$) Given the $o,er "8 in this $aer
g$cine is "rotonated and its movement is conse*uent$ retarded) !he com-ined e##ect o# these t,o
ions generate a region o# high e$ectric #ie$d strength that "ro"e$s the movement o# the negative$
charged denatured "roteins) !he stac.ing ge$ can -e omitted ,hen uti$ising a gradient ge$ given the
continuous$ decreasing "ore si7e rendered - the ge$ /4)
A$ternative$ some researchers common$ uti$ise the native &AG' or nondenaturing &AG') !he
main di##erentiating #actor -et,een these t,o &AG's $iesin that the "roteins ,i$$ on$ -e se"arated
- si7e in the nonnative &AG' ,hi$e the ,i$$ -e se"arated - -oth the "rotein>s native charge and
hdrodnamic si7e as the migrate to,ards the "ositive$ charged anode) !he greater the negative
charge the #aster the "rotein ,ou$d move) !his is concurrent$ im"eded - the sieving e##ect o# the
ge$ that retards the movement o# $arger and -u$.ier "roteins) !he "rotein?s native charge is de"endent
on its amino acid com"osition and su-se*uent " in the running -u##er)
Given that the "roteins ,i$$ sti$$ -e in their #o$ded con#iguration native &AG' "rovides the advantage
o# -eing a-$e to stud the e##ects o# -iochemica$ "rocesses that a$ters "rotein con#iguration or invo$ves
"rotein aggregation) Besides its nondenaturing "ro"erties ena-$e researchers to recover or e+tract
"articu$ar -ands o# the se"arated "roteins #or #urther ana$sis/5
) sua$$ these "roteins retain their active con#ormation and en7matic #unctions /6) 8o,ever on$ nonnative &AG' "rovides the a-i$it
o# mo$ecu$ar ,eight estimation through crosscom"arisons ,ith a $adder)
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Protein Ladder
Goh X.R. and Lim Y.F.Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus
gallus domesticus and Salmo salar using gelelectrophoresis and !estern "lot
n this ana$tica$ stud the use o# :estern -$ot ,as c$ose$ #o$$o,ed) A#ter trans#erring the se"arated
"roteins onto a suita-$e mem-rane anti-odies are designed to detect genera$ o# s"eci#ic "roteins)
!hese mem-ranes t"ica$$ #a$$ under a se$ection o# nitroce$$u$ose or "o$vin$idene #$uoride
constituents) More recent$ mmo-i$on@FL &F Mem-rane - 3igma A$drich has -een o"timised
to increase sensitivit and #$uorescence a""$ications /)
!xperimental o"er"ie#
Four sam"$es o# "re"re"ared e+tracted "roteins #rom Saccharomyces cerevisiae, Torenia sp., Gallus
gallus domesticus and Salmo salar ,ere se"arated ,ith 33 "age and trans#erred onto a
&o$vin$idene #$uoride %&F( mem-rane) !he amount $oaded into the "o$acri$amide ge$ #or the
33 &age ,as uni#ied at 20Cg #or each sam"$e) !he mem-rane ,as then treated ,ith anti-odies
s"eci#ic to 83&0 %0.( and !u-u$in %50.( in a :estern B$ot and #ina$$ ,as visua$ised via
co$orimetric method %,ith !MB so$ution( or chemi$uminescent method) !he co$orimetric resu$ts ,ere
visua$ised #rom Xin Rong and Yi Fei?s &F mem-rane ,hereas the chemi$uminescent resu$ts ,ere
visua$ised #rom Dadia e Rong and Eing Yee?s &F mem-rane) !he ommassie B$ue staining
resu$ts #or the "o$acri$amide ge$ ,as inc$uded #or com"arison as ,e$$)
!he resu$ts #or co$orimetric visua$isation revea$ed signi#icant $osses o# "roteins during running o#
33&AG' and over$oading o# "rotein sam"$es ,hereas the chemi$uminescent resu$ts sho,ed a
certain e+tent o# "rotein sam"$e $oss) Both methods success#u$$ visua$ised the se"aration o# 83&0
and !u-u$in #rom the "rotein e+tract o# a$$ #our organisms)
R!SU$TS
%ig &' olorimetric result ($eft) and hemiluminescent result (right) for proteins extractedfrom plant* fish* chic+en and yeast
Organismolorimetric hemiluminescent
,S ./ Tubulin ,S ./ Tubulin
lant er #aint -and er #aint -and Faint -and er #aint -and
%ish Degative -and Dorma$ -and Faint -and Dorma$ -and
hic+en Degative -and er #aint -and Faint -and er #aint -and
0east Dorma$ -and er #aint -and Fiant -and er #aint -and
Table &' Summary of Results fro ,S ./ (./+D) and Tubulin (1/+D)
DISUSSION
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Goh X.R. and Lim Y.F.Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus
gallus domesticus and Salmo salar using gelelectrophoresis and !estern "lot
!he "resence o# -ands in the 0. and the 50. region a$though ver #aint #or some indicates that
83& 0 and the !u-u$in "roteins ,ere success#u$$ e+tracted and se"arated #rom the "$ant #ish
chic.en and east "rotein sam"$es) From this it can -e assumed that the ma;or ste"s o# :estern
B$otting the trans#er o# se"arated "roteins #rom ge$ to &F mem-rane the -inding o# anti-odies to
83& 0 and !u-u$in "roteins and the generation o# co$oured or $uminescent "roducts #rom en7me
con;ugated anti-odies ,ere success#u$ des"ite "rotein $osses in the "rocess)
Be$o, is the descri"tion #or the :estern B$ot -ands ana$sis #or -oth visua$isation methods
2and
roperties!xplanation
3ery
faint 4
5bsent
n the co$orimetric resu$t the -ands in #irst $ane %"$ant( ,as so #aint that on$ the right
edges ,ere s$ight$ visi-$e) !he $ast #e, -ands o# the $adder ,ere a$so not visi-$e) !he
chemi$uminescent resu$ts a$so sho,ed ver #aint -ands #or the #irst t,o $anes)
!his cou$d -e an indication o# e+treme$ $o, "rotein concentration) rossre#erencing
,ith the oomasie -$ue stain "er#ormed a#ter the 33 &age revea$ed that the #aint -and
,as not a resu$t o# errors during trans#er to mem-rane or anti-od -inding during
,estern -$otting) 3ince the "rotein sam"$es has satis#actor ie$ds as tested "revious$
and the sam"$es ,ere $oaded in the same amount %20Cg( ,e can conc$ude that the error
arose right -e#ore the 33 &age ,as run and not as a resu$t o# ine##icient e+traction in
"rior e+"erimentations)
A "ossi-$e #actor is the #act that the "rotein ,as $e#t in the $oading ,e$$ #or *uite a ,hi$e
-e#ore the ge$ ,as run due to errors in sncing ,ith other grou"s) !his cou$d have
caused the "rotein sam"$es to di##use into the -u##er so$ution -e#ore the ge$ ,as run) t
"rovides an e+"$anation #or the #act that the #irst t,o $anes the $adder and the "$ant "roteins ,ere e+treme$ #aint ,hereas the $ast #e, $anes did not e+"erience such an
e##ect $i.e$ -ecause the #irst t,o $anes ,ere $oaded #irst and e+"erienced more
di##usion) !his e+"$ains ho, across $anes -ands can range #rom indication o# ver high
"rotein $oad %negative -ands #or 83&0 in Fish "roteins( to $itt$e to no "rotein $oad
%e+treme$ #aint -ands #or 83&0 in &$ant "roteins( des"ite the #act that ,e "$aced in
the same amount %20Cg() t ,as h"othesised that on to" o# the $oading se*uence
sma$$er "rotein mo$ecu$es di##use out o# the $oading ,e$$ #aster than $arger mo$ecu$es)
!his e+"$ains the a-sence o# -ands #urther do,n the $adder and the #aint -ands #or
near$ a$$ !u-u$in "roteins in contrast ,ith the 83&0) !he same "ro-$em occurred ,ith
the chemi$uminescent resu$ts)
n the #uture it shou$d -e c$ear$ indicated that a$$ 33 &ages need to run immediate$
a#ter the sam"$e is $oaded and #or students to coordinate #or that "ur"ose)
!xtra
bands
t ,as c$ear that a$though the anti-od ,as meant #or 83& 0 and !u-u$in more than
t,o -ands ,ere o-served in each "rotein sam"$e $ane es"ecia$$ #or the
chemi$uminescent resu$t %the -ands in co$orimetric resu$t ,ere "ro-a-$ $ost ,hen
smeared() <ther than the nons"eci#ic -inding o# anti-odies this cou$d -e the resu$t o#
sam"$e degradation and "rotein over$oading ,hich are more $i.e$ to -e the case)
&revention inc$ude o"timising the use o# "rotease inhi-itors in -u##ers as ,e$$ as
"$acing $ess "rotein $oad in ,e$$s #or "rotein sam"$es ,ith ver high concentrations
%H15CgIC$()
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Goh X.R. and Lim Y.F.Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus
gallus domesticus and Salmo salar using gelelectrophoresis and !estern "lot
Table 6' Western 2lot band analysis
O$ORI7!TRI R!SU$TS 5N5$0SIS (8in Rong* 0i %ei)
<n to" o# the issues a-ove the co$orimetric resu$ts sho, severe smearing and #ro,ning e##ect) <ther
than "rotein degradation a $i.e$ #actor #or smearing is over$oading o# "rotein sam"$es ,hich cou$d
-e "revented - $oading $ess "roteins into the ge$ e$ectro"horesis $oading ,e$$) ssues ,ith :estern
B$ot -ands s"eci#ic to the chemi$uminescent resu$t are as #o$$o,J
2and
roperties
Details
Negati"e For the chic.en and #ish "roteins the -ands a""eared ,hite in the centre and co$oured
at the edges a "henomenon .no,n as negative -ands) !his ,as the resu$t o# su-strate
de"$etion o# the chromogenic su-strate %!MB so$ution( - the -ound anti-odies due toprotein concentration being too high) urious$ this occurred on$ #or #ish and
chic.en des"ite a$$ the $anes containing the same amount o# "roteins)
!o "revent this one cou$d "ossi-$ $oad $ess "roteins #or "roteins ,ith e+treme$ high
concentration re$ative to other sam"$es ,ith disregard to the same "rotein $oad %20Cg(
as used in this e+"eriment)
%ro#ning !he e$ectro"horesis "rocess ,as ran too *uic.$ as a resu$t the "rotein sam"$es at the
edges o# the -and trave$$ed #aster than the main -and "ossi-$ due to sam"$e di##usion
into edges o# $oading ,e$$s ,hich ,ere not "ro"er$ made) A so$ution ,ou$d -e to
investigate the e+"ected running time #or a sam"$e and to ad;ust the vo$tage
according$ ,ith re#erence to the "rogress o# the so$vent #ront movement in the ge$)
Table 6' Western 2lot band analysis for hemiluminescent Results
Furthermore there ,ere "atch "rotein stains near the $adder as ,e$$ ,hich cou$d either -e attri-uted
to uneven distri-ution o# anti-odies as a resu$t o# "$acing more than one mem-rane in a container
during incu-ation) As a resu$t areas o# stri"s in contact ,ith other mem-ranes ma not have as much
anti-oties -ound to the desired "rotein as com"ared to mem-ranes deve$o"ed a$one in a container)
:hat ,e cou$d do is to incu-ate stri"s individua$$ in sma$$er containers to achieve the "ur"ose o#
saving anti-odies and "revent the e##ect o# uneven distri-ution at the same time) !here ,ere "atch "rotein stains near the $adder as ,e$$ ,hich cou$d either -e attri-uted to uneven distri-ution o#
anti-odies as a resu$t o# "$acing more than one mem-rane in a container during incu-ation) As a
resu$t areas o# stri"s in contact ,ith other mem-ranes ma not have as much anti-oties -ound to the
desired "rotein as com"ared to mem-ranes deve$o"ed a$one in a container) :hat ,e cou$d do is to
incu-ate stri"s individua$$ in sma$$er containers to achieve the "ur"ose o# saving anti-odies and
"revent the e##ect o# uneven distri-ution at the same time)
Another "ossi-$e #actor o# the "atch "rotein stains cou$d arise during the trans#er o# "roteins #rom the
"o$acrimide ge$ to the &F mem-rane) :hi$e "rotein trans#er #rom ge$ to mem-rane is a common
source o# error such as un.no,n "rotein -$toches and $oss o# "rotein the #u$$ "ro#i$e o# the "rotein
success#u$$ trans#erred onto the mem-rane is not avai$a-$e #or investigation) 8ence to o-tain *ua$it
trans#ers the &onceau Red de cou$d -e used to visua$ise trans#erred "roteins tem"orari$ to
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Goh X.R. and Lim Y.F.Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus
gallus domesticus and Salmo salar using gelelectrophoresis and !estern "lot
determine i# the "rotein -ands are shar" and distinct or "atch and uneven) ertain "ro-$ems cou$d
arise ,hen ge$s are trans#erred under high vo$tage and tem"erature or ,hen sma$$ "roteins
accidenta$$ trans#er through the mem-rane) :ith the &onceau Red de the trans#er time and vo$tage
can -e o"timised #or a -etter resu$t)
,!7I$U7IN!S!NT R!SU$TS 5N5$0SIS (De Rong* Nadia* 9ing 0ee)
!he -ands #rom this resu$t ,ere more de#ined though the de #ront ,as s$anted) !his ,as "ossi-$
due to -u--$es "resent -et,een the -ottom o# the ge$ and container ,hich a##ects the uni#orm
transmission o# e$ectric #ie$d through the ge$) !here are no signs o# over$oading or the #ro,ning e##ect)
!he se"aration and detection o# !u-u$in and 83&0 #rom the organisms? "rotein e+tract ,as
success#u$ -ut certain -ands ,ere #aint as e+"$ained a-ove)
n genera$ the "oor resu$t #rom co$orimetric detection arose #rom errors during "rotein $oading in the
33&AG' "rocess) o$orimetric detection in genera$ are cost e##ective and sim"$er in "rocedure,here- the a$.a$ine "hos"hatase en7me sim"$ converts the so$u-$e chromogenic su-strate %!MB
so$ution( into co$oured inso$u-$e "roducts ,hich "reci"itates on the site o# conversion and "roduce
co$oured -ands on the mem-rane) hemi$uminescent detection on the other hand uses the horseradish
"ero+idase en7me to convert the "ero+ide I $umino$ so$ution into $uminescent su-strates) As mu$ti"$e
e+"osures are necessar to ca"ture o"tima$ signa$s it is more trou-$esome to use des"ite -eing more
sensitive than the co$orimetric detection method) t a$so cannot -e mu$ti"$e+ed ie) ana$se severa$
"roteins in an assa and ma not -e *uantitative) /9
5$T!RN5TI3!S TO W!ST!RN 2$OTTIN:
mmunoassas remain im"ortant toda as the "re#erred method to detect the "resence andconcentration o# "articu$ar "roteins) :estern B$ot remains high$ s"eci#ic as it se"arates mo$ecu$es
-ased on t,o "arameters "hsica$ si7e and charge hence there is $ess chance #or #a$se "ositive or
negatives) 8o,ever the "rocedures are re$ative$ more com"$e+ and more sam"$e vo$umes are
re*uired #or each run) Limitations such as time consuming "rocedure due to its $o, or medium
through"ut and the #act that it re*uires "roteins to -e denatured made a$ternatives such as 'L3A and
&rotein Assas desira-$e in occasions o# $arge sca$e screenings)
'L3A %en7me$in.ed immunosor-ent assa( uses so$id "hase immunoassas %'A( to detect
antigens in $i*uid sam"$es) 'L3A is ca"a-$e o# testing "rotein mo$ecu$es in its native state and are
technica$$ easier than :estern B$ot %thus "osing a "otentia$ #or automation()
'L3A $i.e :estern B$ot can on$ investigate a $imited num-er o# "roteins at a time) :hen
mu$ti"$e+ ca"a-i$it is needed &rotein Microarras "rove use#u$ as the can ana$se u" to K000
"roteins at a time ,hereas 'L3A can at most ana$se K6 or =96 ,e$$ "$ates and #or :estern B$ot
$ess than 10) Li.e 'L3A &rotein Microarras are use#u$ #or re$ative *uantitation o# "roteins or in
tests that re*uire "roteins to -e in the native stage) !he di##erence $ies in the &rotein Microarra?s $o,
sam"$e usage as ,e$$ as the high through"ut ana$sis) /K
8o,ever ,hen measuring "rotein"rotein interactions it is sti$$ the -est to use :estern -$ot as
'L3A and &rotein Microarras occasiona$$ gives #a$se "ositives or negatives) n short the t"e o#
immunoassa used de"ends on the need and avai$a-$e resources)
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Fig 2. Coomassie Blue staining for extracted proteins
Goh X.R. and Lim Y.F.Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus
gallus domesticus and Salmo salar using gelelectrophoresis and !estern "lot
5!NDI8
R!%!R!N!S
/1 .onomov Me$7er 8 Denov 3toicheva A 3ti$$er 3 and Mann 8 1KKK m"ortance o#
sodium dodec$ su$#ate "oregraduated "o$acr$amide ge$ e$ectro"horesis in the di##erentia$
diagnostic o# Ba$.an ne"hro"ath #rtificial $rgans 590
htt"JII,,,)nc-i)n$m)nih)govI"u-medIKK5019=
/2 !hermo Fisher 3cienti#ic n)d) !ricine "rotein ge$s htt"JII,,,)thermo#isher)comIsgIenIhomeI$i#e
scienceI"rotein-io$ogI"roteinge$e$ectro"horesisI"roteinge$sIs"ecia$i7ed"roteinge$sItricine
"roteinge$s)htm$
/= che Mu$$ins 2002 8o, does an 33&AG' ge$ rea$$ ,or. Mu$$ins La- niversit o#
a$i#ornia 3an Francisco htt"JIImu$$ins$a-)ucs#)eduIsds"ageI
/4 !hermo Fisher 3cienti#ic 2010 !hermo 3cienti#ic &ierce '$ectro"horesis !echnica$ 8and-oo.
v2 htt"sJIItoo$s)thermo#isher)comIcontentIs#sI-rochuresI1601K25'$ectro"horesis8and-oo.)"d#
/5 A$$iance &rotein La-oratories n)d) Dative ge$s htt"JII,,,)a"$a-)comInativege$s)htm
/6 !hermo Fisher 3cienti#ic n)d) <vervie, o# "rotein e$ectro"horesis
htt"sJII,,,)thermo#isher)comIsgIenIhomeI$i#escienceI"rotein-io$ogI"rotein-io$og$earning
centerI"rotein-io$ogresource$i-rarI"ierce"roteinmethodsIovervie,e$ectro"horesis)htm$
/ 3igmaA$drich n)d) mmo-i$on &F Mem-ranes htt"JII,,,)sigmaa$drich)comI$i#e
scienceImo$ecu$ar-io$ogImo$ecu$ar-io$og"roducts)htm$N!a-$e&ageO1456152
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Goh X.R. and Lim Y.F.Separation and analysis of proteins extracted from Torenia sp., Saccharomyces cerevisiae, Gallus
gallus domesticus and Salmo salar using gelelectrophoresis and !estern "lot
/9 Licor Bio B$og :hich :estern B$ot etection Method 3hou$d You seN
htt"JII,,,)$icor)comI-ioI-$ogI,estern-$otting2I,estern-$otdetectionmethod#$uorescence
chemi$uminescenceandco$orimetric
[9] Grace Bio$a-s nd) mmunoassasJ &rotein Arras vs) 'L3A and :esterns
htt"JII,,,)grace-io)comI-$ogI"roteinarrae$isa,esternI
7