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免疫沉淀技术的原理和抗体选择 古朵新闻√
发布时间:2020-01-10   点击次数:20次

    SUIZHESHENGWUJISHUDEFAZHAN,DANBAIZHIYUJIYINZHIJIANDEHUZUOZHUJIANCHENGWEIKEYANRENYUANDEGUANZHUREDIAN,RANSEMIANYICHENDIANJISHUYOUCIERSHENG,BINGBEIYINGYONGDAOGEGEKEYANLINGYUDANGZHONG。BENWENJUJIAORANSEZHIMIANYICHENDIANJISHUDEYUANLI、YINGYONGHEKANGTIXUANZEDENGWENTI,RANGNINDUIQIYOUGENGQUANMIANDELEJIEHERENSHI。

彩搜网SUIZHERENLEIJIYINZUCEXUGONGZUODEJIBENWANCHENG,GONGNENGJIYINZUXUEDEYANJIUZHUJIANCHENGWEIYANJIUDEREDIAN。ERJIYINBIAODADEDIAOKONGYOUSHIGONGNENGJIYINZUXUEDEYIGEZHONGYAOYANJIULINGYU。

彩搜网YANJIUMOUGEDANBAIYINZIDEDIAOKONGGONGNENG,KEYITONGGUODUIDANBAIHUOXING(JIHUOHUOYIZHIQIHUOXING),DANBAISHULIANG(GUOBIAODAOverexpressionHUOJIYINQUEXIANXINGKnockout), YIJIDANBAIGONGNENG(GONGNENGQUEXIANXINGDANBAIDominant-negative mutation)DEKONGZHI,YINGXIANGXIAYOUJIYINDEBIAODA,ERXIAYOUJIYINDEBIANHUAYOUKEYITONGGUOJIYINXINPIAN(cDNA Microarray),YIZHIXIAOJIANZAJIAO(Suppression Subtractive Hybridization),CHAYIXIANSHIRT-PCRDENGFANGFAJINXINGYANJIU。

RANERZHEIXIEFANGFADOUWUFATIGONGZHENGJUZHENGMINGZHEIXIEBIANHUASHISHOUMOUGEDANBAIYINZIZHIJIEDIAOJIEDE,HAISHIJIANJIEDEQITABIANHUADAOZHIDEJIEGUO。SUOYI,YAOXIANGTIGONGDANBAIYINZIZHIJIEDIAOKONGDEZHENGJU,JIUYAOZHIJIEJIANCEDANBAIZHI-DNADEXIANGHUZUOYONG。

CHUANTONGDEFANGFABAOKUOZHUANLUYINZIJIEHESHIYAN(Transcription Factor Assay),DIANYONGQIANYILVBIANDONGFENXI(electrophoretic mobility shift assay),DNase I ZUYINFA(DNase I footprinting),JIAOMUDANZAJIAOXITONGDENG。DANZHEIXIEFANGFADOUYOUYIDINGDEJUXIANXING,BUNENGCHONGFENFANYINGSHENGliQINGKUANGXIADNAYUDANBAIXIANGHUZUOYONGDEZHENshiQINGKUANG,ERQIEHENNANBUZHUODAOZAIRANSEZHISHUIPINGSHANGJIYINBIAODADIAOKONGDEDONGTAISHUNSHISHIJIAN。

彩搜网RANSEZHIMIANYICHENDIANJISHU(Chromatin Immunoprecipitation,JIANCHENGChIP)SHIYANJIUTINEIDANBAIZHIYUDNAXIANGHUZUOYONGDEYIZHONGJISHU。TALIYONGKANGYUANKANGTIFANYINGDETEYIXING,KEYIZHENSHIDIFANYINGTINEIDANBAIYINZIYUJIYINZUDNAJIEHEDEZHUANGKUANG。

彩搜网TEBIESHIJINNIANLAIYOUYUGAIJISHUBUDUANDEFAZHANHEWANSHAN,QIYINGYONGFANWEIYIJINGCONGYANJIUMUDEDANBAIYUYIZHIBAXULIEJIANDEXIANGHUZUOYONG,FAZHANDAOYANJIUMUDEDANBAIYUZHENGGEJIYINZUDEWEIZHIXULIEDEXIANGHUZUOYONG;CONGYANJIUYIGEMUDEDANBAIYUDNADEXIANGHUZUOYONG,FAZHANDAOYANJIULIANGGEDANBAIYUDNAGONGTONGJIEHEDEXIANGHUZUOYONG;CONGYANJIUQIDONGZIQUYUDEZUDANBAIDEXIUSHI,FAZHANDAOYANJIUJIEHEZAIDNAXULIESHANGDEDANBAIFUHEWU。SUIZHEDUIJIYINGONGNENGYANJIUDEBUDUANSHENRU,ZHEIXIANGJISHUZHENGYUELAIYUEDUODEBEIYINGYONGYUKEYANDEGEGELINGYU。

ChIPJISHUDEYUANLI

RANSEZHIMIANYICHENDIANJISHUDEYUANLISHI:ZAISHENGliZHUANGTAIXIABAXIBAONEIDEDNAYUDANBAIZHIJIAOLIANZAIYIQI,TONGGUOCHAOSHENGHUOMEICHULIJIANGRANSEZHIQIEWEIXIAOPIANDUANHOU,LIYONGKANGYUANKANGTIDETEYIXINGSHIBIEFANYING,JIANGYUMUDEDANBAIXIANGJIEHEDEDNAPIANDUANCHENDIANXIALAI。RANSEZHIMIANYICHENDIANJISHUYIBANBAOKUOXIBAOGUDING,RANSEZHIDUANLIE,RANSEZHIMIANYICHENDIAN,JIAOLIANFANYINGDENIZHUAN,DNADECHUNHUA,YIJIDNADEJIANDING。

YINWEIChIPSHIYANSHEJIDEBUZHOUDUO,JIEGUODEZHONGFUXINGJIAODI,SUOYIDUIChIPSHIYANGUOCHENGDEMEIYIBUDOUYINGSHEJIXIANGYINGDEDUIZHAO,ERQIEDUIJIEGUODEFENXIYEXUYAOYOUYIDINGDEJINGYAN。DUIYUGANGGANGKAISHISHIYONGChIPJISHUDEYANJIURENYUANLAISHUO,SHIYONGCHENGSHUDESHANGPINHUASHIJIHEHEXIANGGUANDEJISHUFUWUHUIDADAOSHIBANGONGBEIDEXIAOGUO,BIRUMilliporeGONGSIDEEZ-ChIPSHIJIHEJIUSHIZHUANMENWEICHUXUEZHESHEJIDERUMENCHANPIN。XIAMIANWOMENJIUzuiJIBENDESHIYANBUZHOU,SHIYANZHONGDEXIAOJIQIAOYIJIXUYAOZHUYIDEWENTIJIANDANJIESHAOYIXIA。

1. XIBAOGUDING

彩搜网JIAQUANNENGYOUXIAODESHIDANBAIZHI-DANBAIZHI,DANBAIZHI-DNA,DANBAIZHI-RNAJIAOLIAN,XINGCHENGSHENGWUFUHETI,FANGZHIXIBAONEIZUFENDEZHONGXINFENBU。JIAQUANDEJIAOLIANFANYINGSHIWANQUANKENIDE,BIANYUZAIHOUXUBUZHOUZHONGDUIDNAHEDANBAIZHIJINXINGFENXI。JIAOLIANSUOYONGDEJIAQUANZHONGNONGDUWEI1%,JIAOLIANSHIJIANTONGCHANGWEI5FENZHONGDAO1GEXIAOSHI,JUTISHIJIANGENJUSHIYANERDING。ZHIDEZHUYIDESHI,JIAOLIANSHIJIANRUGUOGUOZHANG,XIBAORANSEZHINANYIYONGCHAOSHENGBOPOSUI,YINGXIANGChIPJIEGUO,ERQIESHIYANCAILIAOYERONGYIZAILIXINGUOCHENGZHONGDIUSHI。JIAOLIANSHIJIANRUGUOGUODUAN,ZEJIAOLIANBUWANQUAN,CHANSHENGJIAYINXING。JIAQUANDEJIAOLIANFANYINGKEBEIJIARUDEGANANSUANZHONGZHI。

2. RANSEZHIDUANLIE

JIAOLIANHOUDERANSEZHIKEBEICHAOSHENGBOHUOMicrococcal NucleaseQIECHENG400~600 bpDEPIANDUAN(YONGQIONGZHITANGNINGJIAODIANYONGJIANCE),YIBIANBAOLUMUBIAODANBAI,LIYUKANGTISHIBIE。CHAOSHENGBOSHISHIYONGJIXIELIDUANLIERANSEZHI,RONGYIYINQISHENGWENHUOCHANSHENGPAOMO,ZHEIDOUHUIYINQIDANBAIZHIBIANXING,JINERYINGXIANGChIPDEXIAOLV。SUOYIZAICHAOSHENGBODUANLIERANSEZHISHI,YAOZAIBINGSHANGJINXING,QIEYAOSHEJISHIDUANSHIXUDECHAOSHENGCHENGXU,BAOZHENGDIWEN。LINGWAI,CHAOSHENGTANTOUYAOJINLIANGSHENRUGUANZHONG,DANBUJIECHUGUANDIHUOCEBI,YIMIANCHANSHENGPAOMO。

ZONGCHAOSHENGSHIJIANYEBUYAOTAIZHANG,YIMIANDANBAIJIANGJIE。Micrococcal NucleaseKEYIJIANGRANSEZHIQIECHENGYIDAOJIGEHEXIAOTI,BICHAOSHENGBOCHULIDEJIEGUOGENGJINGZHI,GENGJUNYI(TU1)。LINGWAI,MEIFANYINGDETIAOJIANBIJIAOWENHE,DUIDNAHEDNA-DANBAIFUHEWUDESUNSHANGJIAOXIAO,ERQIEDANBAIBUYIBIANXING。

MEICHULIRANSEZHISHIYONGYUXINXIANDEXIBAOHUOZUZHIYANGPINHEBINGDONGYANGPIN。ZAIYANJIUZUDANBAISHI,JINGCHANGCAIYONGDEYANJIUFANGFA。YINWEIN-ChIPMEIJINGGUOJIAQUANGUDING,CHAOSHENGBOCHULIHUIDADUANZUDANBAIHEDNADEJIEHE,SUOYIZHINENGXUANZEMEICHULIRANSEZHIDEFANGFA。DUIYUJIAQUANGUDINGDEYANGPIN,YIBANXUANZECHAOSHENGBOCHULIFANGFA。YEYOUYANJIURENYUANSHIYONGMEICHULIDEFANGFAYANJIUJIAQUANGUDINGJIAOWENHEDEYANGPIN。MilliporeGONGSIYOUSHANGPINHUADEMicrococcal NucleaseCHULIDEChIPSHIJIHE(EZ-Enzyme)TIGONG。

RANSEZHIMIANYICHENDIANZHONGDEDUIZHAOYUKANGTIXUANZE

InputDUIZHAO:

ZAIJINXINGMIANYICHENDIANQIAN,XUYAOQUYIBUFENDUANLIEHOUDERANSEZHIZUOInputDUIZHAO。InputSHIDUANLIEHOUDEJIYINZUDNA,XUYAOYUCHENDIANHOUDEYANGPINDNAYIQIJINGGUONIZHUANJIAOLIAN,DNACHUNHUA,YIJIzuiHOUDEPCRHUOQITAFANGFAJIANCE。InputDUIZHAOBUJINKEYIYANZHENGRANSEZHIDUANLIEDEXIAOGUO,HAIKEYIGENJUInputZHONGDEBAXULIEDEHANLIANGYIJIRANSEZHICHENDIANZHONGDEBAXULIEDEHANLIANG,ANZHAOQUYANGBILIHUANSUANCHUChIPDEXIAOLV,SUOYIInputDUIZHAOSHIChIPSHIYANBIBUKESHAODEBUZHOU。

BeadsXUANZE:

彩搜网JIEXIALAI,LIYONGMUDEDANBAIZHIDETEYIKANGTITONGGUOKANGYUAN-KANGTIFANYINGXINGCHENGDNA-DANBAIZHI-KANGTIFUHEWU,RANHOUSHIYONGAgarose beadsHUOMagna beadsCHENDIANCIFUHEWU,TEYIXINGDIFUJIYUMUDEDANBAIJIEHEDEDNAPIANDUAN。ZAIJINGGUODUOCIXIDI,CHUQUFEITEYIJIEHEDERANSEZHIHOU,YONGSDS+NaHCO3XITUOMIANYICHENDIANFUHEWU。Magna beadsSHIJINNIANLAICHUXIANDEYIZHONGXINXINGbeads,TASHIYONGFANGBIAN,BUXIANGAgarose beadsNEIYANGRONGYIPOLIE,SUOYIZAICAOZUOGUOCHENGZHONGGENGJIANDAN,ERQIEMIANQULELIXINDEBUZHOU,JIESHENGBUSHAOSHIJIAN。

KANGTIXUANZE:

彩搜网RANSEZHIMIANYICHENDIANSUOXUANZEDEMUDEDANBAIDEKANGTISHIChIPSHIYANCHENGGONGDEGUANJIAN。YINWEIZAIDANBAIZHIYURANSEZHIJIAOLIANJIEHESHI,KANGTIDEKANGYUANBIAOWEIKENENGYINWEIYUJIEHEWEIDIANDEJULITAIJIN,BUNENGBEIKANGTISHIBIE,SUOYIBUNENGYOUXIAODIZAITINEIXINGCHENGMIANYICHENDIANFUHEWU,ZHIJIEYINGXIANGChIPDEJIEGUO。SUOYIBUSHISUOYOUDEKANGTIDOUNENGZUOChIPSHIYANDE,ZHIYOUJINGGUOChIPSHIYANYANZHENGHOUDEKANGTICAINENGQUEBAOSHIYANJIEGUODEKEKAOXING。

YANGXINGYUYINXINGDUIZHAO:

彩搜网ZAIZUOChIPSHIYANSHI,YIDINGYAOZUOHAOSHIYANDUIZHAO,YINWEIMEIYOUDUIZHAO,HENNANDUISHIYANJIEGUODEKEKAOXINGJINXINGPINGGU。YANGXINGKANGTIHEYINXINGKANGTIDUIZHAOSHIzuiJIBENDESHIYANDUIZHAO。YANGXINGKANGTITONGCHANGXUANZEYUYIZHIXULIEXIANGJIEHEDEBIJIAOBAOSHOUDEDANBAIDEKANGTI,CHANGYONGDEBAOKUOZUDANBAIKANGTIHUORNA Polymerase IIKANGTIDENG。

YINXINGKANGTITONGCHANGXUANZEMUDEDANBAIKANGTISUZHUDEIgGHUOXUEQING。MUDEDANBAIKANGTIDEJIEGUOYUYANGXINGKANGTIHEYINXINGKANGTIDEJIEGUOXIANGBIJIAO,CAINENGDECHUZHENGQUEJIELUN。LINGWAI,HAIYINGKAOLVMUDEDANBAIKANGTIYUDNADEFEITEYIXINGJIEHEDEKENENG,SUOYITONGCHANGHAIHUIXUANZEYIDUIYINXINGYINWU,JIMUDEDANBAIKENDINGBUHUIJIEHEDEDNAXULIE,ZUOWEIGAIKANGTIDEYINXINGDUIZHAO。

zuiJIADEYINXINGDUIZHAOYINWUSHIZAIBAXULIESHANGYOUDEYIDUANYUMUDEDANBAIKENDINGBUNENGJIEHEDEXULIE。RUGUOMUDEDANBAIMEIYOUSHANGPINHUADESHIYONGYURANSEZHIMIANYICHENDIANSHIYANDEKANGTI,ZHIYOUQITAYONGTUDEKANGTISHI,KEYIXIANZUODANBAIZHIMIANYICHENDIAN(Immunoprecipitation)JIANCE。RUGUOKANGTIKEYICHENGGONGDECHENDIANDANBAI,ZAIJINXINGRANSEZHIMIANYICHENDIANSHIYANDEJIANCE。

彩搜网JIAOLIANFANYINGDENIZHUANHEDNADECHUNHUA

彩搜网YONGBUHANDNaseDERNaseHEProteinase K,65 °CBAOWEN6XIAOSHINIZHUANJIAOLIAN,JINGDNACHUNHUAZHUHUISHOUDNAHUOYONGFENlvFANGCHOUTI、YICHUNCHENDIANCHUNHUADNA。DNACHUNHUAZHUCHUNHUADNADEZHILIANGGAO,YOULIYUXIAYIBUPCRDENGFANGFADEJIANCE。YINWEIJIAQUANBUJINJIAOLIANDNA-DANBAIZHI,HAIJIAOLIANDANBAIZHI-DANBAIZHI,SUOYIHAIKEYIDUIDNAXULIESHANGDEDANBAIZHIFUHEWUJINXINGFENXI。ZAINIZHUANJIAOLIANSHIBUSHIYONGProteinase K,RANHOUYONGBINGTONGHUISHOUYOUJIXIANGZHONGDEDANBAIZHI,JINXINGFENXI。

DNADEJIANDING

zuiCHANGYONGDEDNADEJIANDINGFANGFASHIBANDINGLIANGPCRHEReal-time PCR。YOUYUQIDONGZIQUYUDEXULIEJUYOUDUOYANGXINGDETEDIAN,SUOYIBUTONGDEXIBAOXIHUOBUTONGDEDONGWUPINXIDETONGYIJIYINDEQIDONGZIXULIEYOUKENENGBUTONG。ERQIEQIDONGZIQUYUDUOFUHANCGDEXULIE,QIPCRTIAOJIANKENENGXUYAOXIANGYINGDIAOZHENG。YOUTIAOJIANKESHEJIBUZHIYIDUIYINWULAIFANFUYANZHENGChIPSHIYANDEJIEGUO(TU2)。

ChIPJISHUDEYINGYONG

彩搜网RANSEZHIMIANYICHENDIANDEDNASHIYONGYUDUOZHONGFENXIFANGFA。RUGUOMUDEDANBAIDEBAXULIESHIYIZHIDEHUOXUYAOYANZHENGDE,KECAIYONGXIAFENGZAJIAO(Slot blot)DEFANGFA,BABAXULIETEYIXINGTANZHENYURANSEZHIMIANYICHENDIANDEDNAZAJIAO,LAIYANZHENGMUDEDANBAIYUDNABAXULIEDETEYIXINGJIEHE。HAIKEYIGENJUBAXULIESHEJIYINWU,YONGBANDINGLIANGPCRDEFANGFAJINXINGCEDING,HUOCAIYONGReal-time PCRFANGFAJINXINGDINGLIANGFENXI。RUGUOMUDEDANBAIDEBAXULIESHIWEIZHIDEHUOGAOTONGLIANGDE(high-throughput),KECAIYONGSouthernZAJIAO。

DANYINWEIMIANYICHENDIANDEDNALIANGJIAOSHAO,SUOYIZAIYANJIUSHITONGCHANGYAOYONGPCRFANGFAKUOZENGDNATANZHEN,ZAIJINXINGZHENGGEJIYINZUSAOMIAO。HAIKEYIBACHENDIANDEDNAKELONGDAOZAITIZHONG,JINXINGCEXU,XUNZHAOGAIXULIEFUJINDEKAIFANGYUEDUKUANG,FAXIANXINDEJIYINDIAOJIEXULIE。

彩搜网MUQIAN,SUIZHERENLEIJIYINZUCEXUGONGZUODEJIBENWANCHENG,YANJIUMUDEDANBAIHEZHENGGEJIYINZUDEXIANGHUZUOYONGZHUJIANCHENGWEIYANJIUDEREDIAN。YOUYUJIYINZUZHONGDEXINXILIANGFEICHANGDA,SHANGSHUCHANGGUIFANGFATONGCHANGWUFAMANZUKEYANDEXUYAO。JINNIANLAIFAZHANQILAIDEChIP-chipJISHUJIANGJIYINZUDNAXINPIAN(chip)JISHUYURANSEZHIMIANYICHENDIANJISHU(ChIP)XIANGJIEHE,WEIYANJIUMUDEDANBAIYUZHENGGEJIYINZUXIANGHUZUOYONGTIGONGLEKENENG。

ChIP-chip JISHUTONGGUOBIAOJIRANSEZHIMIANYICHENDIANFUJIDEDNAPIANDUAN,HELINGYIGEBEIBIAOJIBUTONGTANZHENDEDUIZHAOZUYANGPINYIQI,YUDNAXINPIANZAJIAO,ZAILIYONGGEZHONGSHENGWUXINXIXUEFANGFADUISHOUJIDAODEXINHAOJINXINGFENXI,JUTIDESHIYANBUZHOUQINGCANKAODr. Richard YoungZAINature ProtocolsSHANGDEWENZHANG。ChIP-chipJISHUYIJINGBEIGUANGFANYINGYONGYUYANJIUZHUANLUYINZIZAIZHENGGEJIYINZUZHONGDEXINHAOWANGLUORANSEZHIXIUSHIJIZHIZAIJIYINZUZHONGDEDIAOKONG,DNADEFUZHI,XIUFUYIJIXIUSHI,JIYINDEZHUANLUYUHEYUNSHUDENGZHUDUOFANGMIAN。

彩搜网RANSEZHIMIANYICHENDIANJISHUHAIKEYONGYUFENXILIANGZHONGDANBAIGONGTONGJIEHEDEDNAXULIE,JIChIP reChIPFANGFA。ChIP reChIPSHIZAIDIYICIChIPDEJICHUSHANGBUJIEJIAOLIAN,ERJIXUJINXINGLINGYIGEMUDEDANBAIDEMIANYICHENDIAN,CONGERDEDAOYULIANGZHONGMUDEDANBAIDOUJIEHEDEDNAXULIE。ZHIDEZHUYIDESHI,YINWEITONGGUOLIANGCIMIANYICHENDIANFUJIDEDNALIANGBIJIAOSHAO,SUOYIZAIFENXISHITONGCHANGYAOBADUOCIMIANYICHENDIANDEDNANONGSUOHOUZAIJINXINGCAOZUO。

JINNIANLAIChIPJISHUYEBEIYONGYUYANJIURNA-DANBAIDEXIANGHUZUOYONG,QIYUANLIYUDNALEISI,YEBAOKUOJIAQUANGUDING,CHAOSHENGBOPOXIBAO,MIANYICHENDIAN,JIAOLIANNIZHUAN,RNACHUNHUAHERNAJIANDINGDENGBUZHOU。SUOBUTONGDESHI,JIAOLIANNIZHUANZHIYONGProteinase K,YAOJINXINGRNACHUNHUAHEBUHANRNaseDEDNaseCHULI,FENXISHIYONGRT-PCR,XINPIANZAJIAOYAOYONGcDNAXINPIANDENG。

彩搜网SUIZHEMIANYICHENDIANJISHUDEYOUDIANYUELAIYUEGUANGSHOUGUANZHU,RUHEGAIJINRANSEMIANYICHENDIANJISHUZHONGDECAOZUOYEYUELAIYUEBEIKEYANRENYUANSUOZHONGSHI。

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