磷酸化蛋白檢測(cè)課件

信號(hào)傳導(dǎo)研究的高級(jí)助手

－高通量，多參數(shù)檢測(cè)磷酸化蛋白磷酸化蛋白檢測(cè)與信號(hào)轉(zhuǎn)導(dǎo)研究的關(guān)系磷酸化作為一種分子開(kāi)關(guān)，能夠動(dòng)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(jí)聯(lián)反應(yīng)將信號(hào)快速傳遞，因而廣泛存在于各種信號(hào)傳導(dǎo)途徑；蛋白磷酸化是細(xì)胞信號(hào)傳導(dǎo)的關(guān)鍵步驟，在細(xì)胞代謝、生長(zhǎng)、增殖、凋亡、癌變和其他生命過(guò)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(guò)磷酸化和去磷酸化改變蛋白活性，借以傳遞信號(hào)，調(diào)控細(xì)胞的生命活動(dòng)。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(cè)的常用方法最常用地方法是利用32P標(biāo)記的無(wú)機(jī)磷酸鹽（32Pi）進(jìn)行生物合成標(biāo)記對(duì)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(shí)驗(yàn)（enzymatickinaseassays）高通量磷酸化蛋白定量檢測(cè)技術(shù)－－液相蛋白芯片技術(shù)磷酸化蛋白多參數(shù)檢測(cè)－－胞內(nèi)流式細(xì)胞術(shù)液相蛋白芯片技術(shù)－BDCBAFlexsetCaptureBeadsLysateSampleDetectorAntibodiesWashAnalyzeonflowcytometer微球和流式檢測(cè)平臺(tái)的強(qiáng)大結(jié)合+100100101101102102103103104104BeadIntensityFL3(670LP)DetectorAbIntensityFL2(585/42BP)微球在流式細(xì)胞儀上的識(shí)別方式ABCDEFGHI123456789TheBD?CBAFlexSet磷酸化蛋白檢測(cè)技術(shù)檢測(cè)細(xì)胞裂解液中的磷酸化蛋白同時(shí)定量分析同一樣本中多個(gè)磷酸化蛋白可根據(jù)實(shí)驗(yàn)需要靈活選擇待檢測(cè)的種類顯而易見(jiàn)的優(yōu)點(diǎn)：實(shí)驗(yàn)操作簡(jiǎn)單，快速；樣本需求量少，不論是檢測(cè)一個(gè)還是N個(gè)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(cè)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(dá)幾十種蛋白同時(shí)檢測(cè)BDCBAandWesternblot檢測(cè)細(xì)胞裂解液中phospho-ERK水平050010001500RELATIVEUNITSOFACTIVITYActivatedcellsControlcellsA431+EGFHeLa+anisoU937+GMCSFMacro+IFN/LPS293+UVHeLa+TNF3T3+osmoticCBAWesternblotBDCBAandWesternblot檢測(cè)細(xì)胞裂解液中phospho-P38水平A431+EGFHeLa+anisoU937+GMCSFMacro+IFN/LPS293+UVHeLa+TNF02500500075001000012500RELATIVEUNITSOFACTIVITYActivatedcellsControlcells3T3+osmoticCBAWesternblot實(shí)驗(yàn)流程準(zhǔn)備并調(diào)試流式細(xì)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(cè)抗體并吸取50ul到對(duì)應(yīng)的試管中分別吸取50ul標(biāo)準(zhǔn)品和待檢樣本到對(duì)應(yīng)的試管中避光室溫孵育4個(gè)小時(shí)加入1ml洗液，離心去上清加入300ul洗液，上機(jī)分析T細(xì)胞受體信號(hào)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(cè)技術(shù)（phosflow)

－單細(xì)胞水平的信號(hào)轉(zhuǎn)導(dǎo)研究單細(xì)胞水平的磷酸化蛋白檢測(cè)的特點(diǎn)結(jié)合了流式細(xì)胞術(shù)的優(yōu)勢(shì)：快速，敏感，定量，多參數(shù)分析；所需細(xì)胞數(shù)少，可以檢測(cè)一些利用傳統(tǒng)方法無(wú)法檢測(cè)的微量細(xì)胞亞群；無(wú)需純化細(xì)胞，可以在最原始的樣本中分析不同細(xì)胞亞群的信號(hào)轉(zhuǎn)導(dǎo)事件，因此為臨床信號(hào)轉(zhuǎn)導(dǎo)的研究開(kāi)辟了一條實(shí)際可行的道路；減少了人為操作帶來(lái)的誤差，結(jié)果更接近機(jī)體的真實(shí)情況可以更為徹底的了解不同的細(xì)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(xì)胞對(duì)于刺激的的反應(yīng)新鮮的全血與放置24小時(shí)的全血中磷酸化信號(hào)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(chǔ)于PBS+1%BSA中的直接熒光標(biāo)記的抗體檢測(cè)分析stainingcontrolsarecrucial.胞內(nèi)磷酸化蛋白檢測(cè)技術(shù)示意圖PermeabilizeStainandAnalyze

IL-4+IL-6刺激全血后檢測(cè)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(shí)驗(yàn)中的藥效監(jiān)測(cè)研究疾病下信號(hào)轉(zhuǎn)導(dǎo)的狀態(tài)以及對(duì)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(duì)于藥物的反應(yīng)，無(wú)需根據(jù)長(zhǎng)時(shí)間的病程觀測(cè)臨床信號(hào)網(wǎng)絡(luò)解析系統(tǒng)生物學(xué):單細(xì)胞信號(hào)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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