信號通路研究相關(guān)技術(shù)(WB、IP、IHC、IF)

SignalingPathwaysResearchTechniquesEdwardLiPh.D.CellSignalingTechnology公司簡介公司目標/CellSignalingResearchToolsAndPlatformsCST主頁ResearchobjectsThroughputProductsorservicesOtherrelatedproductsSignalingnodeorcascadeLowXMT/XPTMantibodyRabbitmAbSignalSilencesiRNAsPathScan?SandwichELISAKits&antibodypairsHTScan?KinaseAssaysPhosphoPlus?AntibodyDuetsSignalStain?IHCKitsConjugatedAntibodiesOrganelleMarkerAntibodySamplerKitCyclicAMPXP?AssayKitCyclicGMPXP?AssayKitStemLite?PluripotencyKitSimpleChIP?KitsCytokines&GrowthFactors/ChemicalModulatorsSignalingpathwayMediumPathScan?AntibodyCocktails&KitsAntibodySamplerKitsPathScan?Multi-TargetSandwichELISAKitsMultipletargetsorsignalingpathwaysHighPathScan?AntibodyArrayKitsHTScan?SubstrateScreeningKitsPathScan?Multi-TargetHCAKitsMotifantibody&PTMScanproteomicproductsandservicesCST產(chǎn)品與研究平臺總覽學(xué)術(shù)資源學(xué)術(shù)資源學(xué)術(shù)資源學(xué)術(shù)資源CellSignalingTechnology?AntibodyValidationTestingInANumberOfApplicationsallowsyoutochoosetheantibodythatworksbestinyourintendedapplication.W=WesternblotIP=ImmunoprecipitationIHC=ImmunohistochemistryIF=Immunofluorescence-basedassaysF=FlowcytometryChIPChromatinImmunoprecipitationCellSignalingTechnology?AntibodyValidationVerifyingSpecificityAndReproducibilityensuresthattheantibodyperformsconsistentlyinallapplicationsspecified.AnalysisofalargepanelofcelllineswithknowntargetexpressionlevelsconfirmstargetspecificityTreatmentofcellswithappropriatekinase-specificactivatorsand/orinhibitorsverifiesspecificityPhosphatasetreatmenttoverifiesphospho-specificityCorrectsubcellularlocalizationortreatment-inducedtranslocationverifiestargetspecificityComparisonofantibodytoisotypecontrolensuresacceptablesignal-to-noiseratioTarget-specificsignalverifiedintransfectedcells,knockoutcells,orsiRNA-treatedcellsBlockingwithantigenpeptideconfirmseliminationofspecificsignalSide-by-sidecomparisonofanewlotwithpreviouslotsensureslot-to-lotconsistencyCellSignalingTechnology?AntibodyValidationIdentifyingOptimalConditionssavesyourprecioustime,samplesandreagents.OptimaldilutionsandbufferspredeterminedPositiveandnegativecontrolcellextractsspecifiedDetailedprotocolsalreadyoptimized內(nèi)容KeyPointsandTroubleshootingsofWesternBlottingIP/ChIPIHCIFKeyPointsandTroubleshootingsofWesternBlottingWhatisWesternblotting?StepbystepguidetotheprocedureandimportantpointsforoptimizationTroubleshootingSummary(whatcommonerrorstolookfor)WesternBlottingProteinsfromcellsareseparatedbasedontheirsizeusinganelectriccurrent.Anantibodytotheproteinofinterestisusedtodeterminewhetherthatproteinispresentinthecells.

ThisisaWesternblot.Thedarkbandsshowwheretheantibodyhasboundtotheproteinofinterest.Youcanseethattheproteinislocatedjustabovethe37kDa“molecularweightmarker”(themeasurementsontheleftsideofthepicture).Thus,the“molecularweight”oftheproteinisapproximately40kDa.WhatisaWesternBlot?WesternBlottingSummaryUsingWesternBlottingToStudyCellSignalingWesternBlottingcanbeusedtoanalyzetheexpression,and/ormodificationstatusofaprotein.Signaltransduction:theprocessofactivatingandcoordinatingcellularresponsestostimuli.Signaltransductioncantakemanyforms.Acascadeofsignalsoffersnumerouspointofsignalamplification,regulation,andpathwayintegration.Kinasecascadesarecommonmechanismswherebyphosphorylationofadownstreamkinaseactivatesit’skinaseactivity,propagatingthesignal.Manysignalingmodifications:cleavage,methylation,acetylation,phosphorylation,ubiquitination,sumolyation,neddylation,farnesylation,…WesternBlottingisonetechniquetoassesspathwayactivity.ABB*CC*DD/C*CascadingPathways&NetworksStepByStepGuidetoProcedureBasicSteps:SamplePreparationSDS(Electrophoresis)Transfer ProbingDetection Phospho-Akt(Ser473)Antibody#9271WesternSampleInHeterogeneousSolutionCellsorTissueSampleSampleSeparation,Probing,andDetection1.Samplepreparation1.Samplepreparation2.Loadingthegel3.Runningthegel4.Transfertothemembrane5.Blocking6.Antibodyincubationanddetection1.Samplepreparation1.SamplepreparationReducinganddenaturing

（還原與變性）CheckdatasheetforadviceSDSdetergent,mercaptoethanol（巰基乙醇）andheatBreaksdisulphidebonds-disruptssecondaryandtertiarystructure

Linearproteinwillthenrunatthecorrectmolecularweight.Mayalsomakeantibodyepitopemoreaccessible.1.Samplepreparation1.Samplepreparation選擇正確的細胞裂解液非常重要WholeCell NP-40(NonidetP-40)orRIPACytoplasmic(soluble)Tris-HClCytoplasmic(cytoskeletalbound) Tris-TritonMembranebound NP-40orRIPANuclear RIPAMitochondria RIPA

(RadioImmunoprecipitationassaybuffer)1.Samplepreparation5×STOPbufferβ-ME1.SamplepreparationSTEP1:SamplePreparationPhysicalDisruption:HomogenizationSonicationistheactofapplyingsound(usuallyultrasound)energytoagitateparticlesinasampleChemicalDisruption:LysisBufferProducts: (formulatedtypicallywithnon-ionicdetergents&withoutglycerol,loadingdye,andreducingagent)CellLysisBuffer(10X)#9803RIPABuffer(10X)#9806ChapsCellExtractBuffer(10X)#9852Laemmli-TypeSDSLysisBuffersProducts: (formulatedwithglycerol,loadingdye,andreducingagent)BlueLoadingBufferPack#7722RedLoadingBufferPack#7723SampleStabilizationProteaseandphosphataseinhibitorsProtease/PhosphataseInhibitorCocktail(100X)#5872PMSF(phenylmethylsulfonylfluoride)1.SamplepreparationSampleStabilization:Proteaseandphosphataseinhibitors1.SamplepreparationSonication（超聲）Centrifugation（離心）Boilingthesampleandputonice,thenstoreat4℃KeypointsinsamplepreparationAvoidmultigelation（避免反復(fù)凍融）Proteinaseinhibitorcocktail（蛋白酶抑制劑）andPMSFRightlysisbufferEnoughresolvesamplewithgrind,homogenizationandsonication（通過研磨、勻漿和超聲使樣品充分溶解）Keypointsinsamplepreparation樣品制備是關(guān)鍵步驟，要求盡可能的獲得所有蛋白質(zhì)，應(yīng)注意以下問題：1：在合適的鹽濃度下，應(yīng)保持蛋白質(zhì)的最大溶解性和可重復(fù)性。2：選擇合適的表面活性劑和還原劑，破壞所有非共價結(jié)合的蛋白質(zhì)復(fù)合物和共價鍵二硫鍵，使其形成一個各自多肽的溶液。3：盡量去除核酸，多糖，脂類等干擾分子。4：防止蛋白質(zhì)在樣品處理過程中的人為的修飾，制備過程應(yīng)在低溫下進行，以避免細胞破碎釋放出的各種酶類的修飾（建議加入合適的蛋白酶抑制劑）5：樣品建議分裝成合適的量，然后冷凍干燥或直接以液體狀態(tài)置-80℃中保存，但要注意不要反復(fù)凍融。1.SamplepreparationProteinConcentrationDetermination（蛋白濃度測定）TheLowryassayTheBradfordassayBCAassayUVabsorbancetomeasureproteinconcentrationMeasurementwithelectrophoresis1.Samplepreparation2.Loadingthegel3.Runningthegel4.Transfertothemembrane5.Blocking6.Antibodyincubationanddetection2.Loadingthegel2.LoadingthegelBackglassplate

(supportsthegel)Spacer

(determinesthicknessofgel)Frontglassplate(supportsthegel,islowerthanbackplatetoallowinnerchamberbuffertocontactgel)Castingstand(supportsgelwhileitpolymerizes)Polyacrylamide

(pouredinasliquidandsolidifiesintoagelmatrixasitpolymerizes)CombPreparingthegelPreparingthegelPreparingthegelPreparingthegelPreparingthegelPreparingthegelPreparingthegelPreparingthegelPreparingthegelPreparingthegelKeypointsinpreparingthegelAllreagentsshouldbeinrightPHandstoredproperly（試劑要有正確的PH值和正確保存）Allapparatusshouldbeclean（保持所有裝置的清潔）APshouldbefreshlyprepared（AP要新鮮配制）Nottoomuchseparatinggel（不要灌太多分離膠）Gelgraduallypolymerize（膠要緩慢凝固）Avoidbubbles（避免氣泡）Keypointsinpreparingthegel1）分離膠不要倒的太滿，需要有一定的濃縮膠空間，否則起不到濃縮效果。

2）上樣蛋白量不應(yīng)超過30ug/mm2(載荷面即：如果你的膠槽是5mm×1mm，則載荷面為：1mm×5mm=5mm2)。

3）gel通常在0.5-1h內(nèi)凝集最好，過快表示TEMED、APS用量過多，此時膠太硬易龜裂，而且電泳時容易燒膠。太慢則說明兩種試劑用量不夠或者系統(tǒng)實際不純或?qū)嵭А?/p>

4）混合攪拌速度太快產(chǎn)生氣泡影響聚合，導(dǎo)致電泳帶畸形。太慢不均勻，特別是甘油。

5）電泳中常出現(xiàn)的一些現(xiàn)象：

︶條帶呈笑臉狀，原因：凝膠不均勻冷卻，中間冷卻不好。

︵條帶呈皺眉狀，可能是由于裝置不合適，特別可能是凝膠和玻璃擋板底部有氣泡，或者兩邊聚合不完全。

拖尾：樣品溶解不好。

紋理（縱向條紋）：樣品中含有不溶性顆粒。

條帶偏斜：電極不平衡或者加樣位置偏斜。條帶兩邊擴散：加樣量過多。2.LoadingthegelLoading(上樣)

20-30ugwholelysateor100ngpurifiedprotein（20-30ug細胞裂解物或者100ng純蛋白）Optimizeamountdependingonexpressionleveloftheprotein（根據(jù)蛋白表達水平來進行優(yōu)化）Loadevenly–sameamountofproteinineachwell（盡量保證每個泳道里的蛋白量保持一致）LoadingthegelLoadingthesamplesLoadingthemarker1.Samplepreparation2.Loadingthegel3.Runningthegel4.Transfertothemembrane5.Blocking6.Antibodyincubationanddetection3.Runningthegel3.RunningthegelAnode-陰極Anegativechargeisappliedtothebufferintheinnerchamber,towhichthetopofthegelisexposedApositivechargeisappliedtothebufferintheouterchamber,towhichthebottomofthegelisexposedCathode+陽極Proteins(whicharenegativelycharged)traveldowntowardthebottom(positive)electrodeTheprogresscanbemonitoredbywatchingthedyetraveltowardthebottomofthegelSmallerproteinsmovemorequicklythroughthegel.ProteinsseparateoutAccordingtosizeProteinsnegativelycharged,movetowardsthepositivecathodeSamplesarepipettedintothewells3.Runningthegel3.Runningthegel所有蛋白樣品調(diào)至等濃度后上樣，樣品兩側(cè)的泳道用等體積的1×loadingbuffer上樣，Marker也用1×loadingbuffer調(diào)整至與樣品等體積低電壓短時間的預(yù)電泳（恒壓10-20V,20-30min），清除凝膠內(nèi)的雜質(zhì)，疏通凝膠孔徑以保證電泳過程中電泳的暢通注意加樣時間要盡量短，以免樣品擴散，為避免邊緣效應(yīng)，可在未加樣的孔中加入等量的樣品緩沖液

凝膠上所加電壓為8V/cm。當(dāng)染料前沿進入分離膠后，把電壓提高到15V/cm，當(dāng)預(yù)染的Marker告訴你，你要分辨的蛋白已經(jīng)到達最佳分辨區(qū)——分離膠的2/3處，OK，電泳結(jié)束了。RunningRunning3.Runningthegel影響跑膠跑的質(zhì)量，有以下幾個因素：1、電壓，小的電壓會使膠的分子篩效應(yīng)得到充分發(fā)揮。電壓越小，條帶越漂亮，濃縮膠80v，分離膠100v就能跑得很好。2、膠的均勻度，膠越均勻，條帶越窄，分離越均勻。倒膠之前，一定要充分混勻，玻璃板一定要干凈，雙蒸水隔離時，一定要比較輕地加上去，避免稀釋上層的分離膠，使膠不均勻。3.RunningthegelLowmolecularweightusehigh%gelHighmolecularweightuselow%gelSlowlyenteringthegel,thenincreasingthevoltage（緩慢入膠，再加電壓）Optimizetherunningtimetoensureproteinswellseparatedbutnotrunoffthegel（慢工出細活）3.Runningthegel1.Samplepreparation2.Loadingthegel3.Runningthegel4.Transfertothemembrane5.Blocking6.Antibodyincubationanddetection4.Transfertothemembrane4.TransfertothemembraneThegelisplacedagainstaspecialmembranethatbindsproteinsNegativelychargedproteinsmoveuptowards+cathodeandontothefilterpaper4.Transfertothemembrane4.TransfertothemembraneCheckthetransfer（檢查轉(zhuǎn)移效果）

ponceaured（麗春紅）,FastgreenFC,CPTS

Coomassiebrilliantblue,indiaink,Amido.black10B

Choiceofmembrane（膜的選擇）:PVDF(Polyvinylidenefluoride)membraneoftengiveslessbackgroundthannitrocelluloseLargermolecularweightproteinsbothtypesoffiltersworkswell.Iflessthan10kDanitrocelluloseshouldbeused-muchhigherbindingcapacity.

（一般情況下選擇PVDF，特別小的蛋白可以選擇硝酸纖維素膜）4.TransfertothemembraneSelectingpropermembraneNC膜尼龍膜PVDF膜靈敏度和分辨率高高高背景低較高低結(jié)合能力80－110ug/cm2>400ug/cm2125－200ug/cm2（適合于SDS存在下與蛋白質(zhì)的結(jié)合）材料質(zhì)地干的NC膜易脆軟而結(jié)實機械強度高溶劑耐受性無無有操作程序緩沖液潤濕,避免氣泡緩沖液潤濕使用前100%甲醇潤濕檢測方式常規(guī)染色，可用放射性和非放射性檢測不能用陰離子染料常規(guī)染色，比較于NC膜，可用考馬斯亮藍染色，可用于ECL檢測，快速免疫檢測。適用范圍0.45um一般蛋白

0.2um一分子量小于20kD蛋白

0.1um一分子量小于7kD蛋白低濃度小分子蛋白、酸性蛋白、糖蛋白和蛋白多糖(主要用在核酸檢測中)糖蛋白檢測和蛋白質(zhì)測序Selectingpropermembrane選擇的根據(jù)主要有：a.膜與目的蛋白分子的結(jié)合能力（也就是單位面積的膜能結(jié)合蛋白的載量），以及膜的孔徑（也就是攔截蛋白的大?。?；b.不影響后續(xù)的顯色檢測（也就是適和用于所選的顯色方法，信噪比好）；c.如果后繼實驗有其他要求，比如要做蛋白測序或者質(zhì)譜分析，還要根據(jù)不同目的來挑選不同的轉(zhuǎn)移膜。PreparingforgeltransferPreparingforgeltransferPreparingforgeltransfer4.Transfertothemembrane分子量(kDa)膠濃度轉(zhuǎn)膜時間（小時)140-2006%2.0-3.0801408%1.5-2.0258010%1.515--4012%0.75<2015%0.5KeypointsintransferringTransferbuffershouldbefreshlypreparedandsupplementedwithmethanol（轉(zhuǎn)移緩沖液現(xiàn)配，加入15-20%甲醇，并冷卻）Avoidbubbles（避免氣泡）Membranesoakinmethanolcompletely（轉(zhuǎn)膜前將膜充分用甲醇浸泡，激活膜表面的正電荷）Marktheuppersurfaceofmembrane(轉(zhuǎn)移后剪角做標記，分清正反面)1.Samplepreparation2.Loadingthegel3.Runningthegel4.Transfertothemembrane5.Blocking6.Antibodyincubationanddetection5.Blocking5.Blocking5%non-fatMilk(脫脂奶粉)inTBSTfor1to2hours.Phosphoproteins,useCasein(酪蛋白)Themembranewillcontaintheproteinsimmobilizedinthesameorientationastheywereonthegel.Themembraneisincubatedwithaproteinblock(usuallyeitherBSAormilk)tooccupythespacesonthemembranethatarenotyetoccupiedbyprotein.Thispreventstheantibodyfromsimplystickingtothemembrane.5.Blocking6.Antibodyincubationsanddetection1.Samplepreparation2.Loadingthegel3.Runningthegel4.Transfertothemembrane5.Blocking6.Antibodyincubationanddetection6.AntibodyincubationsanddetectionHRPSubstrate(底物）egHydrogenperoxide（過氧化物酶）+luminol3-aminophtalate(lightsensitive)EnhancedchemiluminescenceAntigenonmembrane（膜上抗原）Primary（一抗）（二抗）Secondary6.AntibodyincubationsanddetectionTheprimaryantibodyisappliedtothemembraneandallowedtimetobindtotheproteinofinterest.Anyexcessantibodyiswashedaway.Themembraneisincubatedwithasecondaryantibodythatwillrecognizeandbindonlytothefirstantibody.Thisantibodyisconjugatedtoamoleculethatallowsustoseewhereonthemembranetheantibodyisboundoncetheblotisdeveloped.6.AntibodyincubationsanddetectionPrimaryantibody(1XTBS,0.1%Tween-20with5%BSAor5%nonfatdrymilkasindicatedonprimaryantibodydatasheet;)1-2hrRTor4°C

overnightatrecommendedconcentrationSecondaryantibody(WerecommenddilutedinTBSTwith5%nonfatdrymilkand

0.1%

Tween-20)1hoursRTatrecommendedconcentrationKeypointsinantibodyincubationsProperdilution（抗體要合理稀釋）Avoidrepeatuse（抗體避免過度重復(fù)使用）Right2ndantibody（正確種屬的二抗）Properratioof1st:2ndantibody（一抗和二抗的比例要合適）Enoughwashing（充分洗膜，降低背景）6.AntibodyincubationsanddetectionECL

DABFluorescent6.AntibodyincubationsanddetectionFluorescentreagentsaregrowinginpopularityforWesternblottingbecausetheyofferincreasedtimesavingsoverchemiluminescentdetectionandreducedchemicalwaste.Fluorescentapplicationsareoftenmorequantitativethanenzymesystems.Fluorescence-basedWesternblottingrequiresspecialinstrumentation(fluorescenceimager,LI-COROdysseyInfraredImagingSystem).Capturedimagesaresaveddigitally.6.AntibodyincubationsanddetectionSecondaryProducts:6.AntibodyincubationsanddetectionStreptavidin-HRP#3999BiotinylatedAntibodiesPrimaryProducts:Biotinisawater-solubleB-complexvitamin.Biotinbindsverytightlytothetetramericproteinavidin(alsostreptavidinandneutravidin)andisoneofthestrongestknownprotein-ligandinteractions.SecondaryProducts:Streptavidin-HRP#3999Anti-biotin,HRP-linked(D5A7)RabbitmAb#5571Anti-biotin,HRP-linkedAntibody#7075Anti-biotin,AP-linkedAntibody#7055BiotinylatedProteinLadderDetectionPack#7727Biotinisawater-solubleB-complexvitamin.Biotinbindsverytightlytothetetramericproteinavidin(alsostreptavidinandneutravidin)andisoneofthestrongestknownprotein-ligandinteractions.SecondaryProducts:Streptavidin-HRP#3999Anti-biotin,HRP-linked(D5A7)RabbitmAb#5571Anti-biotin,HRP-linkedAntibody#7075Anti-biotin,AP-linkedAntibody#7055Streptavidin-HRP#3999BiotinylatedAntibodiesProtocoloptimizationProtocoloptimizationProtocoloptimizationProtocoloptimizationProtocoloptimizationTroubleshootingNosignal（無信號）Highbackground（高背景）Nonspecificbands（沒有特異條帶）Incorrectbandsize（條帶大小不正確）Whitebandsonblack/greyblot（反片）Patchystaining（顯色不均勻）‘Smear’（條帶模糊）‘Smile’（條帶彎曲或拖尾）Troubleshooting:NOSIGNAL一抗與二抗不匹配選擇針對一抗來源種屬制備的二抗（比如一抗用小鼠來源的，二抗需要選擇兔來源并抗小鼠的二抗）一抗或二抗不足，沒有足夠的抗體結(jié)合到目的蛋白。降低抗體稀釋倍數(shù)，增加抗體濃度，或者延長孵育時間。封閉劑與一抗有交叉反應(yīng)改變封閉劑。Troubleshooting:NOSIGNAL一抗不識別目的種屬的蛋白檢查訂購抗體的說明書，適用范圍內(nèi)是否包含目的種屬；使用陽性對照來檢查抗體質(zhì)量?？乖蛔?/p>

每個泳道加入20-30ug總蛋白;制備樣品要使用蛋白酶抑制物;使用陽性對照.在檢測的組織內(nèi)目的蛋白表達水平低

檢測樣本不表達目的蛋白選擇表達量高的細胞作為陽性對照，用于確定檢測樣本是否為陰性，也可更換細胞系。蛋白轉(zhuǎn)膜效率低利用麗春紅檢測轉(zhuǎn)膜效率;檢查轉(zhuǎn)移裝置看是否電極弄反;轉(zhuǎn)移前膜是否用甲醇浸泡過；轉(zhuǎn)移緩沖液是否忘記加甲醇。Troubleshooting:NOSIGNAL膜過度洗滌減少洗滌時間和強度減少封閉時間和封閉劑濃度。一抗失效使用新配一抗并減少重復(fù)使用疊氮鈉抑制二抗活性。二抗稀釋液內(nèi)不用疊氮鈉顯色試劑盒底物失效。使用新的顯色試劑?？赡艿脑蚣敖ㄗh：●檢測樣本不表達目的蛋白選擇表達量高的細胞作為陽性對照，用于確定檢測樣本是否為陰性?！駲z測樣本低表達目的蛋白提高上樣量，裂解液中注意加入蛋白酶抑制劑?！褶D(zhuǎn)移不完全或過轉(zhuǎn)移可以用麗春紅染膜并結(jié)合染膠（考馬斯亮藍）后確定條帶是否轉(zhuǎn)至膜上或轉(zhuǎn)移過頭；適當(dāng)調(diào)整轉(zhuǎn)膜的時間和電流?！窨贵w不能識別測試種屬的相關(guān)蛋白購買抗體前應(yīng)當(dāng)認真閱讀抗體說明書，確定其是否能夠交叉識別測試種屬的對應(yīng)蛋白?！褚豢狗跤龝r間不足建議4℃結(jié)合過夜?！穸古c一抗不匹配選擇針對一抗來源的種屬的抗體。●洗膜過度洗膜時間不宜過長，加入的去垢劑不宜過強或過多，建議使用0.1%的弱去垢劑Tween-20。Controltable/support/controls.htmlTroubleshooting:HIGHBACKGROUND上樣減少上樣量，以細胞系樣品作為對照轉(zhuǎn)膜選擇合適的膜封閉封閉不充分，延長封閉時間封閉劑濃度優(yōu)化，溶解充分防止封閉劑與一抗之間的交叉反應(yīng)抗體孵育與顯色一抗?jié)舛冗^高，優(yōu)化抗體濃度，盡量使用單抗孵育溫度過高，一抗4度孵育，二抗減少孵育時間和降低濃度其他膜要充分洗滌膜要防止干燥Troubleshooting:HIGHBACKGROUND其他問題●目的蛋白有多個修飾位點（磷酸化位點、糖基化位點、乙?；稽c等），本身可以呈現(xiàn)多條帶。查閱文獻或進行生物信息學(xué)分析，獲得蛋白序列的修飾位點信息，通過去修飾確定蛋白實際大小?！衲康牡鞍子衅渌羟斜静殚單墨I或生物信息學(xué)分析可能性?！駱颖咎幚磉^程中目的蛋白發(fā)生降解加入蛋白酶抑制劑；樣本處理時在冰上操作。MML430kDaTroubleshooting:NONSPECIFICBANDS細胞系傳代過多后蛋白表達譜發(fā)生變化使用未傳代或傳代次數(shù)較少的細胞系進行樣品制備。蛋白本身有很多修飾。

查閱文獻，確定目的蛋白是否存在多種修飾。蛋白被消化或者降解。蛋白樣品確保未被污染，確保含有蛋白酶抑制劑。所檢測蛋白是否存在多種剪接體，導(dǎo)致分子量大小不同。查閱文獻或者通過搜索數(shù)據(jù)庫來確定該蛋白是否存在多種長度不同的編碼mRNA.一抗或二抗?jié)舛雀邥?dǎo)致非特異性條帶優(yōu)化一抗或二抗稀釋濃度。洗滌不充分延長洗滌時間。Troubleshooting:NONSPECIFICBANDS

降解或者還原的可能。確保蛋白樣品未被污染，確保含有蛋白酶抑制劑。

是否存在剪接體。查閱文獻或者通過搜索數(shù)據(jù)庫來確定該蛋白是否存在多種長度不同的編碼mRNA。是否是重組蛋白。Troubleshooting:INCORRECTBANDSIZETroubleshooting:WHITEBANDS/BLACKBLOT太多一抗的時候會發(fā)生這種情況Note–thisisanextremecase!Troubleshooting:PATCHYSTAINING

抗體不足抗體孵育時在搖床上進心，保證膜被抗體充分孵育。

細菌的污染。

抗體避免過久存放和重復(fù)使用。Troubleshooting:‘Smear’我們推薦每個孔道加樣20-30ug使用蛋白酶抑制物加樣過量Troubleshooting:‘Smile’拖尾：樣品溶解不好樣品制備中一定要充分勻漿并超聲裂解。條帶偏斜電極是否不平衡或者導(dǎo)線彎曲條帶向上彎曲凝膠制備不均勻條帶向下彎曲電泳玻璃板之間可能有氣泡Others顯色后發(fā)現(xiàn)條帶間有不均勻的空白點。轉(zhuǎn)膜過程中三明治裝置中有氣泡或者抗體沒有均勻覆蓋膜。在轉(zhuǎn)膜過程中注意防止氣泡并保證抗體總量足夠覆蓋膜雜交結(jié)果中Marker也顯示出條帶?？贵w與Marker內(nèi)蛋白發(fā)生雜交。無妨。條帶顯示不均勻，中空或者弱。凝膠凝固太快導(dǎo)致聚合不均勻。使用合適的促凝劑，在配膠時在膠頂端加上水，防止膜干燥。Immunoprecipitation(IP)Protein:proteininteractionsGeneticapproachYeast2-hybrid(酵母雙雜交)BiochemicalapproachCo-immunoprecipitation（免疫共沉淀）Fusionproteinaffinitychromatography（融合蛋白親和色譜）Cell-biologyFRET-fluorescenceresonanceenergytransfer（熒光共振能量傳遞）ComputationalRosettaStoneCo-regulationPhylogeneticanalysisCo-immunoprecipitation使用針對一個蛋白的抗體去捕獲與該蛋白可能結(jié)合的其它蛋白。在實際應(yīng)用中，以兩個蛋白之間相互作用為例，常常進行正向和反向兩個免疫共沉淀的驗證性分析來確認蛋白之間的相互作用。DeterminationofProtein-ProteinInteractionsImmunoprecipitationofproteinXwithsepharosebeadsProteinextract+SepharosebeadscoatedwithAbstoproteinXXisasingleprotein1DPAGECollectbeadsDissolveproteinsoffbeadsSDSdissolvingbufferXisapartofaproteincomplex1DPAGECo-pptofY&ZwithXProteinxProteinZProteinYCo-immunoprecipitationStepByStepGuidetoProcedure制備樣品樣品的預(yù)純化（preclearing）與目標蛋白抗體共孵育免疫共沉淀清洗Westernblot分析StepByStepGuidetoProcedureSamplepreparationandcelllysisPreclearingofthesolutionIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofinterestWashingoftheantibody-antigencomplexestoremovenon-specificproteinsandcontaminants.Analysisofcomplexes/antigenofinterestbySDS1.Samplepreparationandcelllysis1.a.

裂解液的選擇Detergent-freesolubleproteinlysisbuffer（不帶去垢劑的可溶性蛋白裂解液）Non-denaturinglysisbuffer（非變性裂解液）RIPA(RadioImmunoPrecipitationAssay)buffer(nuclearextracts，核提取物)Denaturinglysisbuffer/bufferfornon-detergentsolubleantigens（變性裂解液）(e.g.Histone，組蛋白)1.Samplepreparationandcelllysis2.temperature(4°C)3.PH4.NaCl150mM5.10%glycerol（甘油）6.ProteaseinhibitorCocktail7.Cation（某些特別的陽離子）,ATP,GTP8.BestfreshlypreparedStepByStepGuidetoProcedureSamplepreparationandcelllysisPreclearingofthesolutionIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofinterestWashingoftheantibody-antigencomplexestoremovenon-specificproteinsandcontaminants.Analysisofcomplexes/antigenofinterestbySDS2.Preclearingofthesolution減少裂解混合物中的非特異性蛋白的含量同時也減少了可能與ProteinA/ProteinG發(fā)生交叉反應(yīng)的蛋白含量。首次進行實驗時最好選擇此步驟，以后熟練后可以跳過。2.Preclearingofthesolution使用正常（兔）血清或者與一抗同一種屬來源的不相關(guān)抗體進行純化。同時加入ProteinA/G純化后一定將血清或者無關(guān)抗體盡量清除StepByStepGuidetoProcedureSamplepreparationandcelllysisPreclearingofthesolution

Incubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofinterestWashingoftheantibody-antigencomplexestoremovenon-specificproteinsandcontaminants.Analysisofcomplexes/antigenofinterestbySDS3.IP:IncubationAntibodyandFormationofAntibody-AntigenComplexes冰上進行操作。100-500μg細胞裂解物同時加入適量一抗?？贵w量及終濃度要通過預(yù)實驗或者查閱文獻和說明書來確定。3.IP:IncubationAntibodyandFormationofAntibody-AntigenComplexes孵育時間通常為3-4小時，或者過夜。通過多次試驗進行優(yōu)化，以降低背景。不要加太多的一抗，否則背景較高。StepByStepGuidetoProcedureSamplepreparationandcelllysisPreclearingofthesolutionIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofinterestWashingoftheantibody-antigencomplexestoremovenon-specificproteinsandcontaminants.Analysisofcomplexes/antigenofinterestbySDS4.Precipitationofthecomplexofinterest

選擇ProteinA或者ProteinG要根據(jù)一抗的來源種屬，或者選擇ProteinA和ProteinG的混合物將槍頭剪成粗口并燒圓，防止加ProteinA/G時損傷磁珠。使用合適的陰性對照一抗，一般選擇與一抗相同種屬的IgG或者無關(guān)抗體。4.PrecipitationofthecomplexofinterestStepByStepGuidetoProcedureSamplepreparationandcelllysisPreclearingofthesolutionIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofinterestWashingoftheantibody-antigencomplexestoremovenon-specificproteinsandcontaminants.Analysisofcomplexes/antigenofinterestbySDS

5.Washingoftheantibody-antigencomplexes充分洗滌很重要不斷增加NaCl的濃度和去垢劑的濃度進行系列洗滌。StepByStepGuidetoProcedureSamplepreparationandcelllysisPreclearingofthesolutionIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofinterestWashingoftheantibody-antigencomplexestoremovenon-specificproteinsandcontaminantsAnalysisofcomplexes/antigenofinterestbySDS6.Analysisofcomplexes/antigenofinterestbySDS由于IP中使用的一抗（針對A蛋白）同時被捕獲并留在復(fù)合物中，在對捕獲蛋白（針對B蛋白）進行煮沸裂解時，可能會導(dǎo)致一抗IgG重鏈（～55kD）和輕鏈（～25kD）與后續(xù)二抗發(fā)生交叉反應(yīng)，這一情況也只出現(xiàn)在A、B蛋白所用一抗相同種屬的情況下。選擇合適的一抗或者二抗，提前考慮。將IP中一抗與磁珠交聯(lián)后再進行洗脫。IPtroubleshooting:Highbackground制備樣品中可能有不完全溶解的大的蛋白復(fù)合體。

制備樣品后進行離心純化，取上清后進行后續(xù)試驗。

洗滌不徹底

多次洗滌，并逐漸增加洗滌緩沖液中的NaCl和去垢劑濃度。

非特異性蛋白吸附于磁珠上。進行預(yù)處理以防止非特異性吸附。IPtroubleshooting:Highbackground抗體本身特異性不好。

選擇合適的抗體，可以考慮單抗。

使用了太多的抗體

進行條件優(yōu)化減少抗體使用了過多的細胞或組織進行裂解。

減少細胞或組織量，我們推薦100-500ug細胞裂解物。IPtroubleshooting:Highbackground太多的非特異性吸附。減少IP時加入的細胞裂解物。對細胞裂解物進行預(yù)處理

抗原降解。

保證樣品中加入了蛋白酶抑制劑，盡量使用新鮮制備的樣品IPtroubleshooting:Highamountofantibodyeluting太多抗體被同時洗脫。

減少抗體使用量，或者將抗體與磁珠交聯(lián)。IPtroubleshooting:Noelutedtargetproteindetected

目的蛋白在樣本中表達量低或者不表達首先對目的蛋白表達量進行檢測，或者加大IP中加入的蛋白裂解物并進行預(yù)處理。抗體使用不足。查閱文獻或者說明書，并多次優(yōu)化或選擇合適的抗體使用量。IPtroubleshooting:Noelutedtargetproteindetected

目的蛋白未被洗脫

保證使用合適的洗脫液，保證洗脫液的強度和PH值合適。

抗體沒有很好的與磁珠相互結(jié)合。

選擇合適的磁珠。

細胞裂解液使用不當(dāng)。

根據(jù)實驗需要選擇，慎重考慮。

ChromatinImmunoprecipitation(ChIP)何為染色質(zhì)免疫沉淀技術(shù)?利用抗原抗體反應(yīng)的特異性，可以真實地反映體內(nèi)蛋白因子與基因組DNA結(jié)合的狀況。應(yīng)用范圍：研究目的蛋白與已知靶序列間的相互作用；研究目的蛋白與全基因組未知序列的相互作用；研究兩個蛋白與DNA共同結(jié)合的相互作用；研究結(jié)合在DNA序列上的蛋白復(fù)合物。ChIP（ChromatinImmunoPrecitation，染色質(zhì)免疫沉淀）

研究研究體內(nèi)蛋白質(zhì)與DNA相互作用的理想技術(shù)。TargetGeneIdentification(individualandglobal)BindingSiteConsensusIdentificationRNA--DNA--ProteinInteractionsAnalysisofEpigeneticPhenomenon(eg,methylation,silencing)ChIP相關(guān)技術(shù)的應(yīng)用ChIP,ChIPsequencing,ChIP-on-chipProDNAChIP是體內(nèi)分析DNA-蛋白質(zhì)相互關(guān)系的技術(shù),能夠真實反映體內(nèi)基因表達調(diào)控的情況電泳遷移率變動分析(EMSA)不足以充分反映生理條件下DNA與蛋白質(zhì)相互作用的真實情況。*體內(nèi)染色質(zhì)高級結(jié)構(gòu)的存在使DNA與蛋白質(zhì)難以接近。*染色質(zhì)結(jié)構(gòu)本身是動態(tài)的*DNA-蛋白質(zhì)相互作用通常是瞬時的為什么要ChIP分析?ChromatinImmunoprecipitation(ChIP)ChIP材料的準備Treatcellswithformaldehyde:protein-proteinandprotein-DNAcross-linksstoptranscriptionfactorsintheirtracks(DAY0)Cross-linkingcanbedoneon:SuspensioncellsAdherentCellsTissuesAnatomicalstructuresSeeprotocolandwebpageforadditionalinformationoncross-linkingandexampleprotocolsProtein-ProteinDNA-DNAOtherOptionsFormaldehydeCrosslinkingDouncingDouncecontainstwosizesAislargeclearanceBissmallclearanceForceofmovingrodBindounceresults:dispersescellclumps“pokes”holesinmembranebreakscellmembranetoreleasenucleiSwellingBufferisusedtobloatcelltoallowcellmembranetobreakmoreeasilyduringDouncingChIP--Day1SonicationSonicationCheckSonicationresultsinmanydifferentsizedfragmentsandshouldbeverifiedforyourexperiment6pulses11pulses21pulses31pulses500bp10kbp3kbpFragmentsizelimitsforPCRPurification100bpAntibodiesareYshapedmoleculeswithbindingsitesforspecificantigens.Theheavychaincancomeinseveraldifferentclasses.HundredsofAbstotranscriptionfactorsarecommerciallyavailableImmunoprecipitation(DAY1&2)Step1:ShowreactivityonaWestern(denaturedepitope).Step2:ShowIPability.PerformIP,runWesternandre-probewithsameAb(IP-Western)Step3:Roughquantification,truebandshouldbe>50%ofcombinedsignalfromotherbands(ENCODEguidelines)ChIPAntibodyValidationPositive1oAb=anti-wheatgermRNAPolII;murineMAb(DAY1)2oAb=RabbitAnti-MouseIgG(DAY2)Negative1oAb=IgGpoolfromrabbitserum(nonspecific,cheap)(DAY1)Additionof2oAbtomatchthesamples(antimouseisnon-reactivetorabbitIgG),someprotocolswillomit(DAY2)StaphAbindsmoreefficientlytoRabbitIgGPositiveandNegativeAbsPositiveControl:mouseanti-PolIIprimarywithrabbitanti-mouseIgGsecondaryNegativeControl:NonspecificRabbitIgGwithrabbitanti-mouseIgGsecondaryThesupernatantfromtheNegativeControlwillbeusedas“Input”later!1°2°2°2°AntibodiesandDNAWashesWashing(DAY2)AdditionofNaHCO3causesantibodiestoreleasetranscriptionfactors(DAY2)0.2MNaClat67ofor3hoursormorereversescrosslinks,DNAfragmentsnowfreeinsolution(DAY2)RNaseAisaddeddigestRNAinsample(DAY3)PCRPurificationkitisusedtopurifyDNAforPCR,comparingpositiveAb,negativeAb,andInput(DAY3)ElutionandCrosslinkReversalPolIIPrimersDHFR3’UTRPrimersVerifythat: