广东11选5

优化Western Blotting的技巧

DANBAIZHIYINJIBUSHIYIMENJINGQUEDEKEXUE,TEBIESHIZAIFANGFALUNYIYISHANG,ZHIJIEHUODE**DE,CHUBANWUJIUXUDEJIEGUOBINGBUSHICHANGTAI。DANSHI,NINKEYICAIQUYIXIEBUZHOULAIYOUHUAWesternYINJISHIYAN,BINGQUEBAOTAMENNENGGOUYI**JIAFANGSHIKAISHI。ZHEIZHONGWEIDIAOGUOCHENGCHENGWEIYOUHUA,WesternYINJIFANGANDEJIGEJIEDUANKEYICONGZHONGSHOUYI。

1. SDS-PAGE

广东11选5 NINDEDANBAIZHIYINJISHIYANZAOZAININHUODEMOZHIQIANJIUKAISHILE。NIKEYIZHUISUDAOYANGPINZHIBEI,DANWEILEKONGJIANDELIYI,RANGWOMENCONGYANGPINFENLIKAISHI。TONGGUOZHEIGE,NIKEYIDEDAONIFANGRUDEDONGXI - RUGUOMEIYOUYIGEHAODENINGJIAO,NIJIANGWUFADEDAOYIGEHAODEMO,TAJIANGCONGNEILIXIANGXIALUOXUAN。RUGUONIZIJITUSHANGNINGJIAO,YAOTEBIEZHUYIQUEBAOHUAZHUANGDEJUNYUNXING。XUANZEZHENGQUEDEBINGXIXIANANBAIFENBI(CANJIANProteintechDE  Western blotXIEYI [PDF])。TONGGUODIKANGZAIGAODIANYAXIAYUNXINGNINGJIAODEYOUHUOBINGSHIYONGDENGXIAOTIJIDE1xYANGPINHUANCHONGYETIANCHONGKONGKONG,BIMIANRANSEQIAN“WEIXIAO”XIAOYING。

广东11选5 **ZHONGYAODESHI,JIARUJUNYUNLIANGDEDANBAIZHI; TONGGUOCEDINGQUEDINGDANBAIZHINONGDU,BINGZHUNBEIJIANSHAONINDEZHUANGZAILIANG,RUGUONINHUODE“TIAOWEN”YINJI。ProteintechYANZHENGSHIYANSHIFAXIAN,MEIGEYONGDAO30μgDANBAIZHITONGCHANGHUICHANSHENGWUTIAOWENHELIANGHAOFENLIDETIAODAI。

2.膜

广东11选5 NINDEMOXUANZEKEYIDUIWesternYINJISHIYANDEJIEGUOCHANSHENGJUDAYINGXIANG。ZHUYAODELIANGZHONGMOLEIXINGSHIPVDFHEXIAOSUANXIANWEISU,DANXIANZAIXIAOFEIPINSHICHANGSHANGYOUJIZHONGBANBEN,BAOKUOLIRUZHENDUIJIYUYINGGUANGDEMIANYIJIANCEYOUHUADEMOHUODIFENZILIANGDANBAIZHI。

PVDFYINQIRENXING,WENDINGXINGHEDUIDADUOSHUSUANHEJIANDENAISHOUXINGERSHOUDAOZHONGSHI(ZHEIYIWEIZHETASHINEIXIEXIWANGBOLIHEZHONGXINTANCEYINJIDERENDE**MO)。PVDFMOHAITIGONGBIXIAOSUANXIANWEISUGENGHAODEDANBAIZHIBAOLIU。

XIAOSUANXIANWEISUMOYIYUDUSAI,TONGCHANGKETIGONGGAOXINZAOBI。DANSHI,NIJIANGWUFABOLIHEZHONGXINTANCETAMEN。BINGZHUYIXIAOSUANXIANWEISUMODEKONGJING。

**HOU,GUANYUSHIYANSHICHUANTONGDEYIJUHUA:BUYAOHAIPAYINSHIYANSHIGUIFANERGAIBIANMOLEIXING。CHANGSHIBUTONGDEDONGXIKENENGSHI**DANBAIZHIYINJIDEGUANJIAN。

3.抗体浓度

JIASHENINYIJINGFUCHULESUOXUDENULILAI  XUANZENINDEKANGTI(ProteintechYETIGONGLEZHINAN),NINDEXIAYIBUYINGGAISHIQUEDING**JIADEGONGZUOKANGTINONGDU。

KANGTIYUKANGYUANZHIJIANDEJIEHESULV(QINHECHANGSHU)SHOUQIZAIRONGYEZHONGDEXIANGDUINONGDU(YIJIQITABIANLIANG,RUWENDUHEpH)DEYINGXIANG。KANGYUANFENGDUTONGCHANGZAIWesternYINJIZHONGGUDING,YINCININTONGCHANGXUYAODIAOZHENGKANGTINONGDUYIHUODEGENGHAODEYINJI。YIYOUZUZHIDEFANGSHIJINXINGCICAOZUO - CESHIJIZHONGXISHIDEKANGTI,TONGSHIBAOCHISUOYOUQITACANSHUBUBIAN - JIANGBANGZHUNINQUEDINGSHIYANDE**JIAKANGTINONGDU。

广东11选5 CIBUZHOUCHENGWEIKANGTIDIDING,YINGZAIMEICISHIYONGXINKANGTIHUOYIZUSHIYANTIAOJIANSHIJINXING。RUGUONINZHUYIDAOXINPICIDEDUOKELONGKANGTIYU**HOUYIPIKANGTIDEBIAOXIANBUTONG,YEYINGGAIZHEIYANGZUO。

RUHEDIDINGKANGTI

XUDUOGONGSIZAIQIKANGTISHUJUBIAOZHONGTIGONGLETUIJIANDEXISHIDU,ZHEIXIETONGCHANGSHIHENHAODEQIUCHANGSHUJU,KEYIBANGZHUNINRUMEN; RANER,RENGRANJIANYIDIDINGKANGTI,YINWEIYONGYUHUODETUIJIANXISHIDUDETIAOJIANKENENGYUNINZIJIDEFEICHANGBUTONG。RUGUOCHANPINSHUJUBIAOJIANYISHIYONG1:1000DEXISHIDU,QINGCHANGSHI1:250,1:500,1:1000,1:2000HE1:4000DEXILIE。

YIBANQINGKUANGXIA,RUGUONINJIANGTUIJIANDEXISHIYEJIASHANGTUIJIANXISHIYEDELIANGBEIHEYIBANXISHIYE(LINGYICEXISHIYEXISHIYE),NINJIANGCHUYUZHENGQUEDEGUIDAOSHANG。RUGUOMEIYOUTUIJIANDEXISHIYEKAISHI,QINGCHANGSHI1μg/ mlDECHUNHUAKANGTIZUOWEIQIDIAN。QINGJIZHUBAOCHISUOYOUQITAXIEYICANSHUXIANGTONG,LIRUYANGPINLEIXING,FUYUSHIJIAN,XIDISHIJIANHEWENDU。

GUANYUERKANGDESHUOMING

广东11选5 YEXUNIBIXUCHANGSHIBUTONGNONGDUDEERKANG - TEBIESHIZAIHUAXUEFAGUANGJIANCEDEQINGKUANGXIA。HRPBIAOJIDEDIERKANGTIDENONGDUZHIJIEYINGXIANGTIAODAIDEWAIGUAN。HRPMEITAISHAOHUIDAOZHIHUAXUEFAGUANGXINHAODI,GUANGPUHUOGUANGPUQUESHI。LINGYIFANGMIAN,GUOGAODENONGDUHUICHANSHENG“SHAOHUAI”DETIAODAI。ZHEISHIYOUYUCHENDIDEKUAISUHAOJINDAOZHIZHONGJIANMEIYOUXINHAODEDAI。HUAXUEFAGUANGSHIJIFUYUDEZHANGDUYEHUIYOUSUOBUTONG(CANJIANXIAMIANDE#6,JIANCE)。

4.阻塞条件

广东11选5 KEYISHIYONGGEZHONGZUSAIHUANCHONGQU,ZHEIXIEHUANCHONGQUDOUBUSHIYONGYUSUOYOUQINGKUANG。WEIMEIGEKANGTI - KANGYUANDUICHANGSHIJIZHONGZUDUANHUANCHONGYESHIHENZHONGYAODE。QINGJIZHU,JIYURUZHIDEZUDUANHUANCHONGYEHANYOUNEIYUANXINGSHENGWUSU,TANGDANBAIHEKENENGGANRAOXINHAODEMEI。LIRU,HANRUDEHUANCHONGYEHANYOUHUOXINGLINSUANMEI,HUIPOHUAININDUILINSUANHUADANBAIZHIDEJIANCE; ZAIZHEIXIEQINGKUANGXIA,CHANGSHITIDAIPIN,RUNIUXUEQINGBAIDANBAIJIZUZHIJI。

5.洗涤

ZAIDADUOSHUQINGKUANGXIA,MEIDANGWOYOUGAOBEIJINGXINHAOSHI,XIUGAIXIAYICIGUANCHUANDEXIDIBUZHOUYIBEIZHENGMINGSHIYOUYONGDE。TONGCHANG,WOTONGGUOJIANGTUWEN-20NONGDUCONG0.05%ZENGJIADAO0.1%LAISHIXIANZHEIYIDIAN。NINHAIKEYISHIYONGYIXIARENHEYIZHONGCELVELAIGAIBIANXIDIQIANGDU:ZENGJIAXIDISHIJIANHETIJI,ZHIXINGEWAIDEHUANCHONGYEGENGHUANHUOSHIYONGGENGQIANGDEXIDIJI(LIRU,SDSDAITITUWEN20)。

6.检测

JIANCEJIEDUANDUIYUHUODELIANGHAODEWesternYINJIXINHAOSHIBUKEHUOQUEDE。YOUXUDUOHUAXUEFAGUANGSHIJIHEHUAXUEFAGUANGTIDAIPIN,RUYINGGUANGJIANCE - SUOYIRUGUONIMUQIANDEJIANCEFANGFABUFUHESHIYANSHIBIAOZHUN,YOUHENDUOXUANZE。LIRU,NINKEYIXUANZEJUYOUGENGGAOLINGMINDUDESHIJIHUOCHANSHENGGENGCHIJIUXINHAODESHIJI。HOUYIZHONGXUANZEJIANGBUKEGULIANGDIZENGJIAYINJIDEZAIXIANXING。

ZAIGENGJIANDANDESHUIPINGSHANG,HUAXUEFAGUANGJIANCEKEYITONGGUOJIAOPIANPUGUANGSHIJIANJINXINGYOUHUA。ZHEIKENENGSHI**CHANGZHIXINGDEYOUHUABUZHOU,YINWEITAKUAISUERJIANDAN。SHIZHONGJIANGNINDEMOSHUIBAOLUZAIYIXILIEDANDUDESHIJIANDIAN(DANQINGJIZHU,DAYUE10DAO15FENZHONGHOU,SUOYOUHRPDIWUJIANGYONGWAN)。NIDEDIANYINGXUANZEYEHUIYOUSUOZUOWEI。WOZONGSHIFAXIANZHUANMENYONGYUHUAXUEFAGUANGJIANCEDEJIAOPIAN - ERBUSHIRENHEBIAOZHUNDEFANGSHEMINGANJIAOPIAN - SHI**JIAXUANZE。

SUIRANYINGGUANGCHENGXIANGXITONGKENENGSHANGWEIGONGMEIGESHIYANSHISHIYONG,DANNINSUOZAIJIGOUKENENGHUITIGONGHEXINHUOZHONGXINSHESHILAITIGONGCIJISHU。JIYUYINGGUANGDEJIANCEXITONGDEZHIJIEYOUSHISHITAMENNENGGOUTONGSHIJIANCEDUOZHONGKANGTI,WUXUBOLIHEZHONGXINTANCENINDEYINJI。

HUOZHE,NINKEYIWANQUANTIAOGUOYIJIKANGTIDEERCIJIANCE,BINGJIANHUANINDEWesternYINJIFANGAN,SHIYONGHRPJIEHEDEYIKANGSHIBIECHANGJIANDESHANGYANGDUIZHAOHEDANBAIBIAOQIAN。(YIXIALIECHUDEXIANGGUANCHANPINJUNYICIGESHITIGONG。)

YOUHUADEZHUYAOMUDESHIZENGJIATEDINGBODUANDEXINZAOBIBINGJIANSHAOFEITEDINGBODUAN(RUGUOCUNZAI)。BUKEFOUREN,RUGUONINXUANZEYOUHUASHANGSHUSUOYOUBIANLIANG,ZEXUYAOHUAFEIDALIANGSHIJIAN; RANER,JISHISHIYILIANGCIGAIDONGYEKENENGYOUSUOBUTONG,CONGZHANGYUANLAIKANSHIJISHANGKEYISHENGSHIJIAN。ZONGDELAISHUO,WORENWEIYOUHUASHIYIXIANGFEICHANGYOUJIAZHIDEGONGZUO - RUGUONIXIANGHUODECHUBANPINZHIDEXIFANGYINJI,NEIMEZHEISHIYIXIANGBIYAODECELVE。