Western Blot Analysis

7/21/2019 Western Blot Analysis

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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)



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

2





!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$()

3





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

4





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)

5



Fig 2. Coomassie Blue staining for extracted proteins



5!NDI8

R!%!R!N!S

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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

6

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ikonomov%20V%5BAuthor%5D&cauthor=true&cauthor_uid=9950183

http://www.ncbi.nlm.nih.gov/pubmed/?term=Melzer%20H%5BAuthor%5D&cauthor=true&cauthor_uid=9950183

http://www.ncbi.nlm.nih.gov/pubmed/?term=Nenov%20V%5BAuthor%5D&cauthor=true&cauthor_uid=9950183



http://www.ncbi.nlm.nih.gov/pubmed/?term=Stoicheva%20A%5BAuthor%5D&cauthor=true&cauthor_uid=9950183


http://www.ncbi.nlm.nih.gov/pubmed/?term=Stiller%20S%5BAuthor%5D&cauthor=true&cauthor_uid=9950183

http://www.ncbi.nlm.nih.gov/pubmed/?term=Mann%20H%5BAuthor%5D&cauthor=true&cauthor_uid=9950183


http://www.ncbi.nlm.nih.gov/pubmed/9950183

http://mullinslab.ucsf.edu/sds-page/

http://www.ap-lab.com/native_gels.htm

http://www.sigmaaldrich.com/life-science/molecular-biology/molecular-biology-products.html?TablePage=14561752


http://www.ncbi.nlm.nih.gov/pubmed/?term=Melzer%20H%5BAuthor%5D&cauthor=true&cauthor_uid=9950183



http://www.ncbi.nlm.nih.gov/pubmed/?term=Stiller%20S%5BAuthor%5D&cauthor=true&cauthor_uid=9950183


http://www.ncbi.nlm.nih.gov/pubmed/9950183

http://mullinslab.ucsf.edu/sds-page/

http://www.ap-lab.com/native_gels.htm



http://www.ncbi.nlm.nih.gov/pubmed/?term=Ikonomov%20V%5BAuthor%5D&cauthor=true&cauthor_uid=9950183





/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

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http://www.licor.com/bio/blog/western-blotting-2/western-blot-detection-method-fluorescence-chemiluminescence-and-colorimetric


http://www.gracebio.com/blog/protein-array-elisa-western/



http://www.gracebio.com/blog/protein-array-elisa-western/

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