磷酸化蛋白檢測課件

信號傳導(dǎo)研究的高級助手

－高通量，多參數(shù)檢測磷酸化蛋白磷酸化蛋白檢測與信號轉(zhuǎn)導(dǎo)研究的關(guān)系磷酸化作為一種分子開關(guān)，能夠動態(tài)調(diào)節(jié)酶活性及蛋白蛋白相互作用，可形成一系列蛋白次序磷酸化的級聯(lián)反應(yīng)將信號快速傳遞，因而廣泛存在于各種信號傳導(dǎo)途徑；蛋白磷酸化是細胞信號傳導(dǎo)的關(guān)鍵步驟，在細胞代謝、生長、增殖、凋亡、癌變和其他生命過程中都扮演著重要角色。如與MAPK通路密切相關(guān)的EGFR，ERK，p38，Caveolin以及STAT通路相關(guān)蛋白都是通過磷酸化和去磷酸化改變蛋白活性，借以傳遞信號，調(diào)控細胞的生命活動。癌癥、感染、炎癥以及其它疾病往往伴隨著異常的蛋白磷酸化現(xiàn)象，因此，這些磷酸化蛋白也是藥物研發(fā)人員感興趣的靶分子磷酸化蛋白檢測的常用方法最常用地方法是利用32P標(biāo)記的無機磷酸鹽（32Pi）進行生物合成標(biāo)記對蛋白磷酸化的研究一般依靠傳統(tǒng)的Westernblot方法和蛋白激酶實驗（enzymatickinaseassays）高通量磷酸化蛋白定量檢測技術(shù)－－液相蛋白芯片技術(shù)磷酸化蛋白多參數(shù)檢測－－胞內(nèi)流式細胞術(shù)液相蛋白芯片技術(shù)－BDCBAFlexsetCaptureBeadsLysateSampleDetectorAntibodiesWashAnalyzeonflowcytometer微球和流式檢測平臺的強大結(jié)合+100100101101102102103103104104BeadIntensityFL3(670LP)DetectorAbIntensityFL2(585/42BP)微球在流式細胞儀上的識別方式ABCDEFGHI123456789TheBD?CBAFlexSet磷酸化蛋白檢測技術(shù)檢測細胞裂解液中的磷酸化蛋白同時定量分析同一樣本中多個磷酸化蛋白可根據(jù)實驗需要靈活選擇待檢測的種類顯而易見的優(yōu)點：實驗操作簡單，快速；樣本需求量少，不論是檢測一個還是N個磷酸化蛋白，所需樣本量都是一樣的；線性范圍寬，且得到定量結(jié)果，units/ml；檢測靈敏度與Westernblot可比，甚至更好可以自主選擇待檢蛋白，可以多達幾十種蛋白同時檢測BDCBAandWesternblot檢測細胞裂解液中phospho-ERK水平050010001500RELATIVEUNITSOFACTIVITYActivatedcellsControlcellsA431+EGFHeLa+anisoU937+GMCSFMacro+IFN/LPS293+UVHeLa+TNF3T3+osmoticCBAWesternblotBDCBAandWesternblot檢測細胞裂解液中phospho-P38水平A431+EGFHeLa+anisoU937+GMCSFMacro+IFN/LPS293+UVHeLa+TNF02500500075001000012500RELATIVEUNITSOFACTIVITYActivatedcellsControlcells3T3+osmoticCBAWesternblot實驗流程準(zhǔn)備并調(diào)試流式細胞儀準(zhǔn)備待檢樣本梯度稀釋標(biāo)準(zhǔn)品混勻并稀釋捕獲微球吸取50ul到試管中稀釋PE標(biāo)記的檢測抗體并吸取50ul到對應(yīng)的試管中分別吸取50ul標(biāo)準(zhǔn)品和待檢樣本到對應(yīng)的試管中避光室溫孵育4個小時加入1ml洗液，離心去上清加入300ul洗液，上機分析T細胞受體信號轉(zhuǎn)導(dǎo)途徑LckZAP-70PItkPMAPKKKPIKKNF-kBRasGrb2SOSRacPPPLCPPDAGPKCPVAVSLP-76PLATPPPPPPPPPPPPRasMEKERKJNKp38MKK4,7MKK3,6MEKK1-4ElkJunATF2MEKK4LATPPRSKPPKineticsofJurkatcellactivationwith

anti-CD3/CD28110100FOLDINCREASEINUNITS/ML05101520TIME(MINUTES)RSKJunLATItkZAP-70PLCp38JNKERKTcellreceptorsignalingresponsestotreatmentwithanti-CD3/CD28antibodiesCellswereactivatedwithanti-CD3/CD28for2minutes.SDSwasaddedtoafinalconcentrationof1%andthematerialwasplacedinaboilingwaterbathfor5minutes.DatainMFIunits.EffectofPD-98059treatmentonTcellreceptorsignalingresponsetoanti-CD3/CD28Jurkatcellswerepre-incubatedwithPD-98059(MEKinhibitor)beforebeingactivatedwithanti-CD3/CD28.

BD?CBAFlexSetphosphoproteinassays具有歷史意義的磷酸化檢測技術(shù)（phosflow)

－單細胞水平的信號轉(zhuǎn)導(dǎo)研究單細胞水平的磷酸化蛋白檢測的特點結(jié)合了流式細胞術(shù)的優(yōu)勢：快速，敏感，定量，多參數(shù)分析；所需細胞數(shù)少，可以檢測一些利用傳統(tǒng)方法無法檢測的微量細胞亞群；無需純化細胞，可以在最原始的樣本中分析不同細胞亞群的信號轉(zhuǎn)導(dǎo)事件，因此為臨床信號轉(zhuǎn)導(dǎo)的研究開辟了一條實際可行的道路；減少了人為操作帶來的誤差，結(jié)果更接近機體的真實情況可以更為徹底的了解不同的細胞亞群在免疫防御中的作用Westernblot和PhosFlow技術(shù)的比較AtheoreticalexperimentcomparingWesternblotandflowcytometrywiththreesamplesandaproteinofinterestat1,10,or50copiespercell.Sample2and3lookthesameviaWesternblot,butwhenstainedwithfluorescentlylabeledantibodies,thedifferencesbetweenthesamplesbecomemorerelevant.(Source:P.O.Krutziketal./ClinicalImmunology110(2004)206–221)ControlUnstimulatedIL4/IL6Simulated全血樣本pStat6pStat3PBMCpStat6pStat3樣本的處理改變了細胞對于刺激的的反應(yīng)新鮮的全血與放置24小時的全血中磷酸化信號的比較Humanperipheralbloodwereeitherleftuntreated(openhistogram)ortreated(shadedhistogram)withhumanIL-6(100ng/ml)orIL-4(100ng/ml)for15minutesat37°C.ThecellswerethenfixedusingBDTMPhosFlowLyse/Fixbufferfor10minutesat37°C,followedbyPermBufferIIIfor30minutesonice.ThesecellswerethenstainedwithPEAnti-Stat3(pY705)orPEAnti-Stat6(pY641).Fresh24hour@4oC24hour@RTSTAT3PESTAT6PEPhosflow與經(jīng)典的WesternBlots的比較固定2%多聚甲醛處理10-15min.破膜以酒精/去污劑處理為基礎(chǔ)

染色存儲于PBS+1%BSA中的直接熒光標(biāo)記的抗體檢測分析stainingcontrolsarecrucial.胞內(nèi)磷酸化蛋白檢測技術(shù)示意圖PermeabilizeStainandAnalyze

IL-4+IL-6刺激全血后檢測胞內(nèi)Stat-3/Stat-6磷酸化水平

Phosflow的應(yīng)用及前景高通量藥物篩選及臨床實驗中的藥效監(jiān)測研究疾病下信號轉(zhuǎn)導(dǎo)的狀態(tài)以及對于不同化合物的反應(yīng)性，以期能夠根據(jù)患者的病程多身定制治療方案可以快速得到患者對于藥物的反應(yīng)，無需根據(jù)長時間的病程觀測臨床信號網(wǎng)絡(luò)解析系統(tǒng)生物學(xué):單細胞信號網(wǎng)絡(luò)制圖X-linkedSCIDresultsfromamutationintheinterleukin2receptorgamma(IL2RG)genewhichproducesthecommongammachainsubunit,acomponentofseveralILreceptors.IL2RGactivatesanimportantsignallingmolecule,JAK3.AmutationinJAK3,locatedonchromosome19,canalsoresultinSCID.

ControlJAK3SCIDX-SCID(gc)Black:ControlGreen:IL-2stimcellsKimberlyGilmoreGreatOrmondStreetHospitalSeverecombinedimmunodeficiencypStat5pStat5pStat511ColorFlowCytometryLookingatSignalinginSubpopulationsofCellsPerez&Nolan,NatureBiotechnology,Vol20,p155-162Multi-DimensionalAnalysisClusteringofBiosignature,ClinicalSignificanceSystemsBiology:SignalingNetworkMappingKinase1Kinase11Kinase2Kinase6Kinase3Kinase1Kinase3Kinase1Kinase11Kinase4Kinase9Kinase11Kinase11Surface2p-p44/42p-p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