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BIOLOGY ASSAYS & PROTOCOLS

WesternBlot protocol
Electrophoresis
SDSPAGE protocol
NativePAGE protocol
Agarose gel electrophoresis protocol WesternBlotting:
PCR protocol
TheabilitytotransferproteinsfromSDSPAGEgelstonitrocelluloseorPVDFmembraneshas
WesternBlot protocol
becomeroutineinmostlaboratories.AnimportantearlypaperwasthatofTowbinetal.(Towbin
ELISA protocol
etal1979).Laterstudiesusedotherkindsofmembranes,notablythenylonlikematerialPVDF,
RNA extraction protocol
whichallowedproteinstransferredfromSDSPAGEgelstobesubjectedtodirectpeptidesequencing(Matsudaira,1975).Weare
Reverse transcription protocol
assumingyouranaregularSDSPAGEslabgel(seeSDSPAGEgels).NitrocelluloseandPVDFbothworkwellforblotting
Real TimePCR
nitrocelluloseismorefragile,beingsomewhatbrittle,andisalsohighlyflammable,infactexplosive(anothernamefornitrocelluloseis
guncotton,putamatchtoasmallpieceifyoudon'tbelieveuswecannotbeheldresponsibleifyouburnyourlabdown).
SDS PAGE Protocol
CoIP Protocol WesternBlotRelatedAntibodies:
Western Blot Protocol
ELISA Protocol SecondaryAntibody
H7N9 HA/Hemagglutinin (New)
TagAntibody
LoadingControlAntibody
IsotypeControlAntibody

WesternBlotProtocol:
1.Rungelasusual.Takegeloutofelectrophoresisapparatus.CutintosegmentsasrequiredPartofgelcanbestaineddirectlyin
CoomassiebrilliantblueR250(2.5gCoomassieBrilliantBlueR250,450mlsmethanol,100mlsglacialaceticacid,waterto1liter).
Parttobeusedforelectroblottingisputintotapwateronshaker,afterfirsthavingmarkeditunambiguouslytoidentifytop/bottom,left
andrightetc.

2.Leaveinwateronshakerfor5minutes.Thisstepcanbesubstitutedbywashingthegelinelectrotransferbuffer(seebelow)for5
minutes.

3.Weuseasemidryblotter,whichwehavefoundtobequicker,moreeconomicalandeasierthanfullysubmergedblottingmethods.
WecutWhatman3Mfilterpaperstothesizeofourgels,andplacethreeoftheseontothesemidryblotter.Thesearethenwetwith
transferbuffer(weroutinelyuse3.03gTrisbase,14.4gGlycine,10%Methanolperliter).Thegelisputontothefiltersandaprewetted
nitrocellulosefilterisputontopofthegel.AlternatelyputaPVDFmembraneontopifyouareusingPVDFrememberitisessentialto
prewetthePVDFin100%methanol.Greatcareshouldbetakentoensurethatnoairbubblesareanywhereinthisstackof
membranes.ThenthreemorewettedWhatman3Mfiltersshouldbeplacedontopofthepile,againtakinggreatcarenottohaveany
bubblesinpile.Putthetopontotheapparatusandscrewitdown.Proteinsintransferbufferarenegativeinchargemostlydueto
residualSDSandtheythereforemovefromveto+vepole.Sothe+veelectrodeisabovethenitrocelluloseandthevesideisbelow
thegel.

4.Runfor30minutesto1hourat~100mA.Themostreliablewayofdoingthisistouseapowerfulpowersupply200500mAandputit
onconstantvoltage,withasettingof5to10Volts.Lowmolecularweightproteins(20kDaorless)willtranserin30minutesat5Volts,
whilehighermolecularweight(150kDaormore)transferin60minutesat10Volts.

5.Afterrunningdisassembletheapparatusandremovenitrocellulosefilter.Stainthisfor5minutesonshakerinPonceaureagent
(0.25%PonceauSin40%methanoland15%aceticacid).DestainwithregularSDSPAGEgeldestainsolution(7.5%methanol,10%
aceticacid).Ifyoutransferredefficiently,theproteinscanbeseenaspalepinkbands.ThistellsyouwhetherthetransferwasO.K.or
notandalsoexactlywherethebandsare.Youcanphotograph,photocopyormarkthepositionofthebandsdirectlywithapencil.If
youcan'tseeanybandsatthisstage,it'sprobablysmarttotrytooptimizesteps3and4.Thegelmaybediscardedormaybestained
asusualincoomassie,toseehowmuchproteinisleftbehind.

6.AfterPonceaustainingputthenitrocellulosefilterintoblockingsolution,suchas1%bovineserumalbumin(BSA)or1%Carnation
nonfatmilk(NFM),for20minutesto1hratRTor37C.SincetheNFMworksjustaswellasBSAbutismuchcheaper,thereisreally
nogoodreasontouseBSA.Ponceaustainingwillfadetobecomecompletelyinvisible.Carryonwithantibodyincubationsetc.

AntibodyIncubations:
1.Putinantibodysolutions.Volumeshouldbeenoughtocoverblotandallowittofloatfreelywhenyouagitate.Ininitialexperiments,
antibodyconcentrationshouldgenerallybeabout1:1001:1,000forascites,CL350tissueculturesupernatantorantiserum,undiluted
to1:10formonoclonalsupernatant,andabout110g/mlforapureIgG.Ifdilutionbringsantibodyconcentrationtolessthan50gs/ml,
addsomeBSAorNFMtoactascarrierprotein(e.g.makeBSAorNFMconcentration1mg/ml).Incubateforatleast1hourwithshaking
(canberoomtemperatureorat37C,canalsodoovernightat4C).

2.WashmembranesinTBS(10mMTris,154mMNaCl,pH=7.5plus0.1%Tween20)for3timesatleastfiveminuteseachtimewith
extensiveagitation.

3.Incubateinsecondantibody(peroxidaseconjugate,phosphataseconjugateorradioactive).AddBSAorNFMcarrierasbeforeif
necessary.Incubateforatleastonehouratroomtemperatureor37Ccanalsodoovernightat4Cwithshakingasbefore.

4.WashmembranesinTBS(10mMTris,154mMNaCl,pH=7.5plus0.1%Tween20)for3timesatleastfiveminuteseachtimewith
extensiveagitation.

AlkalinePhosphataseBlotSystem
1.Incubateinalkalinephosphataseconjugatedantibodyagainsttheprimaryantibody(e.g.Goatantimouse,rabbitorchickenbuy
fromSigmaorsomeothertrustedsource).Typicalconcentrationis1:1,000inTBS(10mMTris/HCl,154mMNaCl,pH=7.5).Addasmall
amountofBSAorNFMtoactascarrier.Incubatefor1houratroomtemperature(or37C)withshaking.

2.WashinTBSthreetimes5minuteseach.(N.B.thealkalinephosphataseenzymeisinhibitedbyEDTA,whichchelateszincand
magnesium,andbyphosphate,whichinhibitsforwardreaction.MakesurethereforeyouuseTBSwhichisEDTAandphosphatefree
Don'tmakeupdeveloperinPBS!)

3.Putintodeveloper.Bufferis100mMTris/HCl,100mMNaCl,5mMMgCl2 pH=9.5.To10mlofthisadd33lBCIPT(5bromo4chloro
3indolylphosphate,ptoluidinesalt,makeup50mg/mlinwaterorDimethylformamideinwatermakesayellowsuspension)and33l
ofNBT(NitroBlueTetrazolium,also50mg/mlinwater).Canstorethesesolutionsat20C.Canbuythissolutionmadeupalreadyfrom
 Sigma.Reactionproductispurple,andappearsinafewminutescanincubateforuptoanhourifthesignalisweak.Watch
developmentofreactionandstopwithwater.Someofbackgrounddisappearsondrying.

HorseRadishPeroxidaseStaining
AfterwashingofblotsinTBSorPBS(mustnothaveazideinwashbuffer!Thisinhibitstheperoxidaseenzyme)addreactionmixture.
Thisis20mls0.1MTris/HClpH=7.2(Vectastainbuffer).200lNiCl(80mg/ml),6l30%hydrogenperoxide,1mlof5mgs/ml
diaminobenzidine.(Weargloves,DABiscarcinogenic).AlternateprotocolMake20mlsammoniumacetatebuffer(50mM,pH=5.0).
Add1mlof10mg/mlDiaminobenzidine,40l30%hydrogenperoxide.Brownreactionproductisseenin110minutes,notquiteso
niceasabovemethod.

ChemiluminescenceStaining
Chemiluminescencehasanadvantageofperhapsanorderofmagnitudegreatersensitivitythanthedyebasedmethodsabove.In
addition,severalfilmsmaybeexposedfromasingleblot,givinganadvantageininterpretationofweakandstrongsignalsonthe
samemembrane.Howeveritrequiresadarkroomtoperformandismoreexpensiveinreagents.Reagentsaregenerallyboughtina
kit,andwerecommendsimplyfollowingthekitinstructions.

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