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如何获得定量western blot数据 古朵新闻√
发布时间:2019-07-04   点击次数:210次

YUSUOYOUDEJIANCEFANGFAYIYANG,western blotSHUJUDEZHILIANGHEDINGLIANGSHIZAIJIANCEGUOCHENGZHONGBORUO、BIANHUADUODEHUANJIE。ZHUNQUEDUHEZHONGXIANXINGCHADEBUZHOUJIANGXIANZHIwestern blotDINGLIANGCHENGDU。XIAOBIANTIGONGLESHIYONGDESHIYANCAOZUODEJIANYI,GUANYURUHEHUODEGAOZHILIANGDE、DINGLIANGDEwestern blotSHUJU,YIBIANNINKEYIDECHUWENDINGDESHIYANJIEGUO。

1.DINGLIANGDEDINGYI

ZAITAOLUNRUHECONGwestern blotZHONGHUODEDINGLIANGSHUJUZHIQIAN,XIANDINGYIYIXIAWOMENSUOSHUODE“DINGLIANG”SHISHENMEYISISHIHENYOUYONGDE,HEWEIDINGLIANG,BIXUFUHEXIALIEZHUNZE:

? 使用一个定义的过程进行测量,即western blot
? 这个过程产生一个可重复的结果,即是度
? 测量反映了一个“真实”的结果,即是准确度

Western blotGUOCHENGXUYAOMINGQUEDINGYIYIJIJINKENENGBAOCHIYIZHIDEBUZHOU,SHUJUXUYAOKEZHONGFU,BINGQIESHUJUYINGGAIYUSHIYONGZHENGJIAOFANGFASHOUJIDESHUJUYIZHI,LIRUZHIPUFENXIFA。

彩搜网ZAIBENWENZHONG,WOMENJIANGZHONGDIANTAOLUNRUHEMANZUZHEIXIEBIAOZHUN,QIANGDIAOZAINAXIEDIFANGXUYAOTEBIEZHUYIYIZHIXINGHUOZHIXINGSHIJIYANZHENGHEZHIKONG,KEYIHUODEwestern blotKEKAOXINGHEZHONGXIANXINGDESHUJU。

2.使用相同的方法处理样品

YIZHIDEYANGPINZHIBEIHECHULISHICHANSHENGDINGLIANGwesternSHUJUDEZHONGYAODEBU。SHENZHISHIFUYUSHIJIAN、bufferCHENGFENHUOWENDUSHANGDEWEIXIAOCHAYI(KEBIANDESHIWEN,ZAI-20℃LENGDONGYUZAIBINGSHANGBAOCUN),YEHUIXIANZHUJIANGDIwestern blotSHUJUDEZHONGXIANXING。YIDANJIANLILEKEKAODEXIBAOHUOZUZHILIEJIEHEYANGPINCHULIFANGAN,ZAIZHUNBEIXIANGHUBIJIAODEYANGPINSHI,YINGJINKENENGDIZUNXUNGAICHENGXU。

3.KANGTIYANZHENG

QIKANZAZHIXUYAOYIXIEKANGTIYANZHENGDEXINXI,SHISHISHANG,DUIYUJIANCEGANXINGQUDANBAI,ZHEISHIYIGEHENHAODESHIYANJISHU,ZAINIZIJIDESHIYANSHILIYONGNIZIJIDEYANGBENHEJISHULAIYANZHENGKANGTI。ZHEIZAISHIYONGYIQIANCONGWEIFABIAOGUODEKANGTISHIYOUQIZHONGYAO。

JINJINYANZHENGNIDEKANGTIJIANCEDAODANBAIZAIYUQIFENZILIANGSHIBUGOUDE。ZHONGYAODESHIYAOBIAOMINGJIEHESHITEDINGYUMUBIAODANBAIDE,zuiHAOTONGGUODUOZHONGFANGFA。GUOJIKANGTIYANZHENGGONGZUOZUYIJINGFABULEYITAOHENHAODEJIANYI,QIZHONGLIECHULEWUZHONGBUTONGDEKANGTIYANZHENGFANGFA。QIZHONGSIZHONGSHIYONGYUYANZHENGWestern blottingZHONGSHIYONGDEKANGTI,WOMENZAIZHEILIZONGJIELEZHEIXIEYANZHENGFANGFA。

? 基因验证:通过敲除基因或通过RNAi消除目标蛋白的翻译,通过显示蛋白表达信号被消除或减少来证明抗体的特异性。
? 正交验证:证明使用抗体测量蛋白丰度与使用质谱法等正交方法测量蛋白丰度有很大相关性。
? 独立抗体:证明使用你自己的抗体测量蛋白丰度与使用第二种已验证的抗体测量蛋白丰度密切相关,该抗体可以识别同一目标蛋白上不同的、不重叠的表位。
彩搜网? 标记蛋白表达(这是独立抗体方法的一种特殊变体):表达一个带有附加表位标签的靶蛋白,并指出使用你自己抗体测量蛋白丰度与使用识别表位标记的验证抗体测量蛋白丰度相关性。

QINGZHUYI,KANGTIYANZHENGSHITEDINGYUYINGYONGCHENGXUDE——WEIELISAsYANZHENGDEKANGTIBUWEIwestern blottingYANZHENG(FANZHIYIRAN),YINWEIJIEHEFASHENGZAIMINGXIANBUTONGDETIAOJIANXIA。

彩搜网4.YANZHENGXINHAOXIANXING,YOUHUADONGTAIFANWEI

 

WEILECONGwestern blotZHONGHUODEZHUNQUEDECELIANGJIEGUO,NINXUYAOQUEBAOJIANCEDAODEXINHAOYUDANGQIANDEDANBAILIANGCHENGZHENGBI,HUANJUHUASHUO,JIANCEZAIXIANXINGFANWEINEI。RANER,ZAIwestern blottingGUOCHENGZHONGYOUDUOGEBUZHOUKEYIDAOZHIXINHAOBAOHE,YINCIXUYAOZAIMEIGEBUZHOUZHONGYANZHENGXINHAODEXIANXING。ZHUYI,QIZHONGXUDUOBUZHOUHAIKEYIYOUHUA,YIDAXIANDUDITIGAOXINHAODEDONGTAIFANWEI。

YANZHENGSUOSHIYONGDEYANGBENLIANGZAIXIANXINGFANWEINEI

WEILEHUODEWENJIANDEDINGLIANGDANBAIYINJISHUJU,WOMENJIANYISHENGCHENGYITIAOBIAOZHUNQUXIAN,HANGAININJIANGPINGGUDEQUANBUYANGPINLIANG,CESHIMEIGELIANGDEDUOGEZHONGFUYANGPIN(TU1)。XINHAOQIANGDUYUYANGBENLIANGDEGUANXITUJIANGBIAOMINGNINKEYIJIAZAIDUOSHAODANBAI,BINGQIERENGRANKEYIQUEXINNINDEXINHAOCHUYUXIANXINGFANWEINEI。

QINGZHUYI,YINGQUEDINGMEIGEKANGTI - DANBAIDUIYINGDEXITONGXIANXINGFANWEI。

 

JIANCHAZHUANYINTIAOJIANDUIXINHAOXIANXINGDEYINGXIANG

彩搜网MONENGRONGNADANBAIDESHULIANGKENENGBAOHE。WOMENJIANYICESHIJIGEZHUANYINSHIJIAN,BINGXUANZEKEXINGCHENGXIANXINGXINHAODEDUANSHIJIAN。RUGUOZAICESHIDESUOYOUZHUANYINSHIJIANXINHAODOUBAOCHIBAOHEZHUANGTAI,NEIMEHENKENENGSHIQITADEBUZHOUDAOZHILEXINHAOBAOHE。ZAIZHEIZHONGQINGKUANGXIA,KEYIJIXUYOUHUAQITACANSHU,RANHOUFANHUIZHUANYINSHIJIAN,YANZHENGMORONGLIANGSHIFOUBAOHE。

DIDINGKANGTI

彩搜网WOMENJIANYIZHENDUININQIWANGJIANCEDEQUANBUBADANBAILIANGCESHIBUTONGDEKANGTIXISHIDU。 ZHEIJIANGQUEBAONINDEJIANCELINGMINDUZUYIJIANCEDILIANGDEMUBIAODANBAI,BINGQIEJUYOUZUGOUKUANDEDONGTAIFANWEI,KEJIANCEDAOzuiGAOLIANGDEDANBAIRENGCHUYUXIANXINGFANWEINEI(TU2)。


图2.滴定一抗,寻找zui佳稀释倍数。在10 mL Azure化学发光封闭缓冲液中
彩搜网加入三种不同量的一抗,其他western blot条件不变。zui佳抗体加入量是5 μL。

YOUHUATUXIANGCAIJISHIJIAN

彩搜网PINGGUXIANXINGDEHOUYIBUSHIYINJIDAOJIANCEXITONGDEPUGUANGSHIJIAN,WULUNSHIJIAOPIANHAISHISHUZICHENGXIANGYIHUOSAOMIAOYI。 DOUXUYAOYOUGEHELIDEPUGUANGSHIJIANBAOZHENGNENGJIANCEDAORUOXINHAODANBAITONGSHIQIANGXINHAODANBAIHAIBUGUOPU,RENGCHUYUXIANXINGFANWEINEI。

彩搜网XUANZEHESHIDEDIWUYEYONGYUDINGLIANGHUAXUEFAGUANGwestern blot

彩搜网TONGGUOSHIDANGDEZHIKONGHEZHENGQUEDEECLDIWUHUAXUEFAGUANGYEKEYIDINGLIANG。XUANZEYIGEDONGTAIFANWEIGUANG,LINGMINDUGAO,BANSHUAIQIZHANGDEDIWUYEJINXINGKEKAOHEKEZHONGFUDEDINGLIANG。

彩搜网DONGTAIFANWEISHOUJIANCEJISHUDEYINGXIANG

JIAOPIANDEDONGTAIFANWEITONGCHANGZAI1 logFANWEINEI,ZHEISHIYIGEXIANGDANGZHAIDEDONGTAIFANWEI。XIANGBIZHIXIA,PMT(GUANGDIANBEIZENGGUAN)DEDONGTAIFANWEIKEYIZAIDAYUE6.3log。

 

 

5.ZONGDANBAIGUIYIHUA

彩搜网GUIYIHUADUIYUKEKAODEwestern blotDINGLIANGSHIZHIGUANZHONGYAODE——TONGGUOZHAODAOYIGEQIWANGZAIBUTONGYANGPINHETIAOJIANXIABAOCHIBUBIANDELIANG,NINKEYIZHIKONGYANGPINZHIBEI、SHANGYANGHEZHUANYINDEBIANHUA,BINGSHENGCHENGMUBIAODANBAIDUIGAIBUBIANTEZHENGDEXIANGDUIZHI。

YIZHONGGUANGFANSHIYONGDEFANGFASHISHIYONGKANJIADANBAIJINXINGGUIYIHUA,LIRUGAPDH, tubulinHE actin,QIBEIRENWEIJUYOUWENDINGQIEBUBIANDEBIAODASHUIPING。 JINDEYIXILIEYANJIUBIAOMINGZHEIZHONGJIASHEYOUHENDUOQUEXIAN,XIANZAIXUDUOMIANYIYINJIZHUANJIAHEKEXUEQIKAN,RUJournal of Biological ChemistryZAZHI,JIANYISHIYONGZONGDANBAIJINXINGGUIYIHUA。ERBUSHIKANJIADANBAIZHI,CHUFEIYONGYUGUIYIHUADEKANJIADANBAIJINXINGWENDINGBIAODAYANZHENG2,4,5,6


彩搜网6.使用一致的western blot协议

ZIXIYANGEDIZUNXUNJIDINGFANGANYOUZHUYUJIANSHAOBIANYIXINGBINGTIGAOCELIANGDU,CONGERSHIXIANWENJIANDEDINGLIANGDANBAIYINJI。 NINBUJINYINGGAITONGGUOSHIYONGXIANGTONGDESHIJIHUOSHISHIPICIDUIZHAOLAIJIANSHAOBIANYIXING,BAOCHIFUYUHEXIDISHIJIANXIANGTONGYEYOUZHUYUQUEBAOYIZHIHEKEKAODEJIEGUO。

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