學(xué)習(xí)小禮包-3實(shí)驗(yàn)相關(guān)

Westernblot實(shí)驗(yàn)步驟與注意事項(xiàng)by妍兒&逛吃逛吃逛 WBprotocol&注意事項(xiàng)by七 WB測組織和細(xì)胞的蛋白表達(dá)量by威 蛋白提取相關(guān)知識介紹by 膜蛋白提取介紹by BCA蛋白濃度檢測（碧云天P0012為例）by常春 半干轉(zhuǎn)與濕轉(zhuǎn)對比by劉 WB實(shí)驗(yàn)細(xì)節(jié)by雷 問題條帶的心得體會by WesternBlot常見問題及對策byWBtrouble byo&慧 做好WesternBlotting，你需要知道的那些事兒by阿拉 WB數(shù)據(jù)處理與圖表制作by阿波沒有 WB寫作by巧巧、梁鸞&Westernblot實(shí)驗(yàn)步驟與注意事項(xiàng) by妍兒&逛吃逛吃逛吃對分別用RIPA或LaemmliSampleBuffer液提取的蛋白進(jìn)行Westernblot，電轉(zhuǎn)緩沖液、封閉液、TBST/PBST洗膜液、抗體、預(yù)染的ProteinMarker、TBST：TBS（10×）100mL+純水900mL+Tween205mLPBST：PBS（10×）100mL+純水900mL+Tween205mL封閉液：脫脂奶粉/BSA（牛蛋白）5g+TBST溶解定容至100ml后4℃壓縮膠一般選用5%左右的濃度，不同KDa的蛋白應(yīng)選擇合適濃度的分離膠 （一）SDS電玻璃板（提前完成）：一只手扣緊玻璃板，另一只手取少量洗衣粉輕輕配5%的濃縮膠，加入TEMED后立即搖勻后灌膠。將剩余空間的蛋白原液量，并將各樣本蛋白原液蒸水配至相同的蛋白濃度。取1.5mL離心管，加入一定量的5×SDSloadingbuffer以及四倍體積的配好的電泳：先選擇80V恒壓電泳，待溴酚蘭遷移至分離膠后(約)，提高電酸談補(bǔ)充材料 移液器吸取1×電泳液各孔，保證無氣泡蓋上蓋子（當(dāng)只電泳一塊膠時，取出另一半電泳槽內(nèi)膠板夾轉(zhuǎn)一張膜需準(zhǔn)備2張10×10cm海綿、2張7.0×8.0cm的濾紙和1張7.5×8.5cm將已跑好的SDS凝膠平鋪在PVDF上選擇轉(zhuǎn)印程序

RunTime 20Vfor1min23Vfor25Vfor

1h 檢測完一個分子后，用Stripbuffer洗滌15min、再重新用TBST/PBST洗根據(jù)抗體說明書用1×iBind溶液稀釋抗體，一抗二抗稀釋后總體積要達(dá)吸取5mL的1×iBind在卡片中間區(qū)域再加入1mL1×iBind溶液，中間區(qū)域形成液團(tuán)蛋白面朝下將PVDF膜覆蓋在液團(tuán)上，低分子量靠近厚端滾輪趕走PVDF膜中氣泡蓋上iBind蓋子，打開孔蓋，有1-4號孔（1）1孔中加入2mL稀釋后一抗（2）2孔中加入2mL的1×iBind溶液（3）3孔中加入2mL稀釋后二抗（4）4孔中加入6mL的1×iBind溶液室溫最少孵育2.5小時，也可4℃過夜孵育孵育完成后，將PVDF膜置于去離子水中漂洗即可進(jìn)行免疫發(fā)光檢測作為催化劑夠提氧促使丙酰單體聚合TMD為加速劑能夠促進(jìn)P氧基，加速丙烯酰胺單體的聚合。空氣溫度較低時TMD使用Bio-Rad或者碧云天的產(chǎn)建議兩邊加入預(yù)染的ProteinMarker，方便觀察不同KDa蛋白的分離情況，也方便后續(xù)的裁膜。的ProteinMarker的品牌和貨號:Thermo26616、延長電轉(zhuǎn)時間，最好是每次電轉(zhuǎn)時都使用新的電轉(zhuǎn)液。不同KDa的蛋白電轉(zhuǎn)封閉常用脫脂奶粉或BSA，建議建議做磷酸化蛋白的western時使用BS（止過亮，阻礙。WBprotocol&注意事 by七WesternBlot蛋白樣品一、貼壁細(xì)白樣品凈將干擾后續(xù)的蛋白濃度測定和westernblot檢測結(jié)果。PMSF（100mM，PMSF合，可在臨用前加，于冰上充解，為使細(xì)胞充解培養(yǎng)瓶要經(jīng)常來于4℃下12000rpm離心20min以去除細(xì)胞碎片（提前開離心機(jī)預(yù)冷的細(xì)胞有時按照實(shí)驗(yàn)要求只能加100-200μ的裂解液，按照上述操作，直接壁細(xì)白樣品的操作外還應(yīng)收集培養(yǎng)液中的細(xì)胞。做法如下：二、懸浮細(xì)白樣品酸談補(bǔ)充材料 充解后，即可用將勻漿液轉(zhuǎn)移至EP管中，然后在4℃下12000rpm離20min，取上清進(jìn)行蛋白定量并分裝，暫時不用的蛋白于-80℃保存一、玻璃使用柔軟的羊毛刷和的玻璃清潔劑將PAGE膠的玻璃板充分刷洗，拿取玻璃板時要戴著干凈套，并且只接觸玻璃板的側(cè)邊，保持玻璃板表面的酸談補(bǔ)充材料 沸了。同時在水中煮蛋白樣品有下面弊端：aEP管容易炸開，樣品丟失。b.慢加入樣品。每個孔加樣體積大致保持一致，空的孔也上同樣體積的電泳跑濃縮膠時用80-100V，使樣品壓成一條線，跑到分離膠以后用200V跑，使不同大小的蛋白分子充分分離轉(zhuǎn)一塊膠需準(zhǔn)備濾紙（多張薄濾紙或2張轉(zhuǎn)膜的厚濾紙和1張膜最后轉(zhuǎn)膜夾能緊緊貼住“濾紙-膠-膜-濾紙三明治”為宜轉(zhuǎn)膜時電極方向注意是膜正膠負(fù)同上方法進(jìn)行二抗孵育，室溫下孵育1h后，用TBST在室溫下脫色搖4次以上，每次5min；進(jìn)行化學(xué)發(fā)光反應(yīng)一般使用HRP-ECL發(fā)光法，操作，制得短一點(diǎn)，一般剛貼上就可以拿開。如果暗室中看不見熒光，就需要延長問題1：轉(zhuǎn)預(yù)處理膜時沒有完全均勻地靶蛋白等電點(diǎn)等于或接近轉(zhuǎn)移緩沖液pH值甲醇濃度過高SDS濃度不夠轉(zhuǎn)移大于100KDa的蛋白時，需要在轉(zhuǎn)膜液中加入適量SDS轉(zhuǎn)移時間不夠問題2：背景膜沒有完全均勻濕透膜被污染封閉洗膜一抗孵育不當(dāng)二抗孵育不當(dāng)過度縮短時間另外，NC膜的背景通常比PVDF膜的背景更干凈，可以選擇使用NC膜問題3：沒有陽性條帶或條帶較樣本中不含目標(biāo)蛋白封閉過度抗體問題酶失活HRP抑制劑干擾延長時間問題5：條帶位置（大?。┎粚?；或有非特異性條蛋白問題抗體問題問題6：背封閉液中有體HRP耦聯(lián)二抗中有體問題7：膜上出現(xiàn)反像（暗背景上白色帶HRP含量過高ECL發(fā)光液進(jìn)行檢測 測組織和細(xì)胞的蛋白表達(dá) by威參考資料=abcam實(shí)驗(yàn)指南+CST實(shí)驗(yàn)指南+takara實(shí)驗(yàn)指南+酸談+威少經(jīng)驗(yàn)總結(jié)+其他實(shí)驗(yàn)指南精品內(nèi)容=目的原理+蛋白質(zhì)樣品+蛋白質(zhì)定量之BCA法+上樣準(zhǔn)備之蛋目的：WB是用特異性抗體對PAGE電泳的蛋白質(zhì)樣品進(jìn)行，并通過分析的位置和深度來獲得特定蛋白質(zhì)在所分析的細(xì)胞或組織中的表達(dá)情白質(zhì)樣品轉(zhuǎn)移到固相載體（PVDF膜orNC膜）上，固相載體以非共價鍵形式也可以直接倒）（若有鼻涕樣的說明RIPA加的不夠，還需要再加，但不可加太多）超聲結(jié)束后，冰上裂解20分鐘超聲結(jié)束后，冰上裂解20分鐘配平）(6)將離心后的上清分裝轉(zhuǎn)移至1.5ml離心管，取一小部分測蛋白濃度，其他進(jìn)loadingbuffer采用1：4稀釋成1×）。金屬?。?00℃，5-10分鐘室溫冷卻：10-20分鐘三、蛋白質(zhì)定量之BCA主要有BCAowry法和Bradford法三種，BCA法是主流，其他具體看說明書配置不同濃度的標(biāo)準(zhǔn)蛋白液：取10μl標(biāo)準(zhǔn)品稀釋到100μl釋到260μl，使最終濃度為0.5mg/ml。第二個梯度：4μl+PBS76μl，其他類提前配置好，多配一孔以防萬一），每個樣品3個復(fù)孔，每孔20μl，加入五、SDS電泳吸干。（威少：緩慢勻速將膠加至距上面1.5cm處，一般到第一個綠配置并加入電泳液：.將之前配好的膠固定在電泳裝置上，加入1×電泳1000ml（配方各個實(shí)驗(yàn)區(qū)都有）后馬上拔梳子，兩3μl，如果兩側(cè)有空的梳孔，應(yīng)該加入20μl1X的loadingbuffer，起“壓邊”作至分離膠時將電壓調(diào)至120V90min，一般在溴酚藍(lán)跑出膠時停止電泳，也加SDS，分子量大的話可以加入0.01-0.03%；貓大版說＜100KDa不用加補(bǔ)充材料 貓大版（威少補(bǔ)充材料 把膠放在膜上面，膜兩邊濾紙不能相互接觸，否則短路。濾紙也有酸 補(bǔ)充材料 濾紙放在下面）個實(shí)驗(yàn)區(qū)，Tween20比較粘稠，可以先將槍頭剪掉一部分，再進(jìn)行吸取。防止一抗或/和二抗與膜的非特異性結(jié)合，需要進(jìn)行膜的封閉）orBSA（一3.封閉：可以用TBST一下，將膜置于盛封閉液的孵育盒（可以淘寶定制(1)配置：用1%BSA（一區(qū)用TBST）液體稀釋二抗（比例看說明書，種屬根據(jù)（二抗作用時間必須嚴(yán)格控制）10min；5min；5min；貓大：5min×812次。仔細(xì)洗滌，否則顯影背景高）(1)底物化學(xué)發(fā)光ECL顯色：在1.5mlEP管（外包錫紙，避光放在4℃）按1:1混將膠片進(jìn)行掃描或拍照，用凝膠圖像處理系統(tǒng)ImageJ分析目標(biāo)帶的分子量 yOne因其高昂的售價令很多人望而卻步，貓大用的是Gel-Proyzer。(1)對WB 去除背景：Process|SubtractBackground 設(shè)置定量參數(shù)：yze|SetMeasurements,點(diǎn)擊Area，MeanGray及IntegratedDensity設(shè)置單位：yze|SetScale,在“unitoflength”的方框里輸入“pixels” 選擇FreehandSelection，盡量把條帶圈起來，點(diǎn)擊鍵盤m，出來IntDen灰 數(shù)據(jù)IntDen進(jìn)行分析 如果條帶不正，需修正，Imagetransformrotate調(diào)節(jié)angle值，直到條帶水 SecondLane（快捷鍵Ctrl+2），最后yzeGelPlotlanes。 （如過夜）洗膜過度：勿過度洗一抗失效：使用新鮮抗體，重復(fù)使用有效濃度會降低二抗受疊氮鈉抑制：避免疊氮鈉和HRP檢測試劑盒過期和底物失活：使用新鮮的底抗體孵育溫度過高：4℃孵育Tween20。脫脂奶粉含有酪蛋白，該蛋白本身就是一種磷酸化蛋白，會結(jié)未結(jié)合蛋白質(zhì)洗滌不充分：增加洗滌次數(shù)膜的選擇導(dǎo)致的高背景：NC膜比PVDF檢測到過的新蛋白或同一蛋白中具有相似表位而結(jié)構(gòu)不同的閉時間和/或溫度，向封閉緩沖溶液中加入0.05%的Tween20，抗體稀釋液抗體質(zhì)量：嘗試使用不同的抗體抗體純化：使用親和純化的抗體，減少非特異條帶(1轉(zhuǎn)膜時膜上有氣泡或抗體在膜上分布不均：轉(zhuǎn)膜過程中盡量去除氣泡，抗體深背景出現(xiàn)白色條帶（非預(yù)期背景細(xì)菌污染：4°C抗體量不足：確保在振蕩孵育時抗體充分浸沒膜蛋白提取相關(guān)知識介 by白淀，通過高鹽的細(xì)胞白抽提試劑抽提得到細(xì)胞白。利用去污劑破壞脂質(zhì)雙分子層，破裂細(xì)胞溶解蛋白蛋白變性使其穩(wěn)抑制蛋白酶活性快速裂解，多種裂解成分經(jīng)過精心優(yōu)化，能快速使細(xì)胞裂以用于蛋白活性檢測（信號傳遞研究和酶動力學(xué)檢測）和WesternBlot等各還可以用于WesternBlotting。適用于培養(yǎng)細(xì)胞（包括懸浮細(xì)胞）和新鮮組細(xì)胞裂解成分一般使用的是表面活性劑，常用的有曲拉通X-100（TritonX-和SDS為離子型。不同裂解成分的裂解強(qiáng)度不同，一般來說TritonX-100和水解。蛋白酶抑制劑（proteaseinhibitor）從廣義上指與蛋白酶分子活性中心上的一些基團(tuán)結(jié)合，使蛋白酶下降，甚至，但不使酶蛋白變性的物質(zhì)。各乙二胺四乙酸（EDTA）：DFP的抑制常數(shù)相似，可以有效抑制胰蛋白酶（trypsin）、糜蛋白酶（thrombin）等蛋白酶，作為PMSF和DFP的替代物，AEBSF的毒性更低物水解酶和氨基肽酶活性的鋅金屬蛋白酶），丙氨酰氨基肽酶（氨肽酶M/胃酶抑素A（PepstatinA）：天冬氨酸蛋白酶可逆抑制劑氟化鈉（sodiμmfluoride）：酸性磷酸酶可逆抑制劑焦磷酸鈉（sodiμmpyrophosphate）：絲氨酸/蘇氨酸磷酸酶不可逆抑制劑β-甘油磷酸（β-glycerophosphate）：絲氨酸/鉬酸鈉（sodiμmmolybdate）：酸性磷酸酶不可逆抑制劑二水酒石酸鈉（sodiμmtartratedihydrate）：酸性磷酸酶可逆抑制劑咪唑（imidazole）Tabam1%Triton1%Triton100,,0.1%1%NP-1%NP-1%NP-1%強(qiáng)中強(qiáng)取對白的取劑是是是是是是是是是是是是WB,IP,Co-WB,WB,WB,IP,WB,IP,Co-WB,四、不同樣本類型的提取方法（以總蛋白為例貼壁細(xì)①倒掉培養(yǎng)液，將瓶倒扣在吸水紙上使吸水紙吸干培養(yǎng)液（或?qū)⑵恐绷⒎泞诿科考?xì)胞（一般需要5*106細(xì)胞）加預(yù)冷的PBS（0.01MpH7.2～7.3）④裂解完后，用干凈的刮棒將細(xì)胞刮于培養(yǎng)瓶的一側(cè)（動作要快），然后；⑤于4℃下12000rpm離心20min。（提前開離心機(jī)預(yù)冷⑥將離心后的上清分裝，于－80懸浮細(xì)微升裂解液的比例加入裂解液。再用手指輕彈以充解細(xì)胞。充解后應(yīng)加藥的貼壁細(xì)①將培養(yǎng)液倒至15ml離心管中，于2500rpm離心②棄上清，加入PBS并用槍輕輕吹打洗滌，然后2500rpm離心5min。棄上③經(jīng)常彈一彈以使細(xì)胞充解④充解后，即可用移液器將裂解液移至1.5ml離心管中，然后在4℃12000rpm離心20min，取上清分裝并置于－80℃保存膜蛋白提取介 by合的非常緊密，一般只有用去垢劑(detergent)使膜解后才可分離出來。相反，外周膜蛋白不磷脂雙層，通常通過與脂質(zhì)的極性基團(tuán)和/或表面的整合以下以某一種提取膜蛋白的試劑盒（ThermoScientificMem-PERPlusMembraneProteinExtractionKit，89842）為例簡介該種提取膜蛋白方法的流用3ml細(xì)胞液細(xì)胞沉淀，300g離心5分鐘移除并丟棄上清液。將細(xì)胞重懸在1.5ml的細(xì)胞液中并轉(zhuǎn)移到2mL的收集細(xì)胞時采用細(xì)胞刮，不應(yīng)采用胰酶消化（貓大老師曾講過）煮沸更容易導(dǎo)致蛋白，物不能有效進(jìn)入膠中。四、使用某試劑盒提取膜蛋白進(jìn)行WB實(shí)驗(yàn)的范例文獻(xiàn)：范例范例BCA蛋白濃度檢測（云天P0012為例） BCA法測定蛋白濃度可以兼容樣品中高達(dá)5%的SDS，5%的TritonX-BCA蛋白濃度測定試劑盒（碧云天P0012）96孔37℃恒溫培養(yǎng)蛋白標(biāo)準(zhǔn)品：5mg/ml釋為0.5mg/mlBSA（Tips：稀釋后的0.5mg/mlBSA蛋白標(biāo)準(zhǔn)可以一次全BCA工作液配將試劑按下表加到96孔板第一列8個標(biāo)準(zhǔn)品孔中，反應(yīng)各孔加入200μlBCA工作液，96孔板震蕩30sec，37℃恒溫培養(yǎng)箱放置30分鐘。（Tips：也可以室溫放置2小時。BCA法測定蛋白濃度時，顏色會一般提取的蛋白濃度應(yīng)該在多少組織在1-30μg/μl，細(xì)胞低一些大概0.1-10μg/μl半干轉(zhuǎn)與濕轉(zhuǎn)對 by劉二、半干轉(zhuǎn)與濕轉(zhuǎn)操作流程對比——流程對比/圖解對比/細(xì)節(jié)對比/條件設(shè)定的一濕轉(zhuǎn)圖半干Westernblot全程操作流程見“酸談”WB實(shí)驗(yàn)細(xì) by雷BCA定量時盡量不使用96孔板最邊緣一圈（由于有些儀器邊緣效應(yīng)配膠時，板子一定徹底干凈問題條帶的心得體 by10min響結(jié)果，可以參考抗體信息及查閱文獻(xiàn)來解決二、WB條帶背景有黑點(diǎn)三、WB條帶顯影無條帶（10KDa四、WB條帶有邊緣規(guī)則白點(diǎn)用袋孵一抗時，未除凈氣泡六、WB條帶歪斜 WesternBlot常見問題及對 轉(zhuǎn)膜液（transferbuffer）、TBST、電泳緩沖液（runningbuffer）或另一個原因可能是脫脂奶粉掩蓋了抗原。在這種情況下使用BSA或減少使高背景通常是由于與PVDF膜結(jié)合的抗體濃度過高造成的也可能導(dǎo)致背景過高。建議試試不同的時間來找到最佳時間WBtrouble o&缺乏信號：染色不均勻：抗體被細(xì)菌污染可導(dǎo)致染色不均勻，4℃保存抗體并使用新微笑條帶：可能的原因是遷移過快或電泳溫度過高（改變了pH值和遷移度），應(yīng)降低遷移速度或低溫電泳（冷庫或冰上）性條帶，只有特異性條帶能被封閉從而。蛋白樣品有可能沒有充分變性，而不是5min，使得蛋白充分做 Blotting，你需要知道的那些事 by阿拉子量蛋白質(zhì)的轉(zhuǎn)膜，使用孔徑0.1或0.2微米的膜。PVDF結(jié)合低分子量膜的選擇主要考慮3點(diǎn)后續(xù)是否有stripandreprobing的打裂，不適合stripandreprobing。PVDF膜結(jié)合蛋白的能力較強(qiáng)，質(zhì)地結(jié)實(shí)，適合stripandreprobing。但疏水性強(qiáng)，使用前需要用甲醇激活，容易干膜。注意不要用未戴手套指接觸膜，最好是用鑷子。手套要帶無粉的。有粉如果打算stripptingandreprobing，切記先洗去原有抗體再風(fēng)干，因?yàn)轱L(fēng)干0.5%的脫脂牛奶（溶解于0.1%Tween的PBS）最常用。脫脂奶粉的品牌酪蛋白（Casein）。常用1%的濃度，但酪蛋白干粉難溶于水進(jìn)行檢測。如果對照中出現(xiàn)條帶，即說明avidin或streptavidin結(jié)合到了內(nèi)源體蛋白的緩沖液中（常用0.1%BSA溶液）。否則大部分抗體會吸附于容器就洗膜液成分而言，標(biāo)準(zhǔn)的洗膜液是PBST/TBST溶液，Tween-20的濃度于0.3%時影響抗體的結(jié)合。Tritonx-100，NP-40或SDS等洗滌劑可洗掉目標(biāo)蛋膜表面的流動。十、STRIPAND在同一張膜上對多種蛋白的表達(dá)進(jìn)行檢測顯然更有效率，所用方法就是常用strip技術(shù)是2%SDS和100mM2-巰基乙醇(2-ME)或二硫代蘇糖醇法有效但會導(dǎo)致大量的目標(biāo)蛋白丟失。另法是在室溫、pH=2的條件下用膜徹底及再次封閉（因?yàn)閟trip也會去除原有的封閉蛋白）。Strip的液浸泡30分鐘，再徹底洗滌)。該反應(yīng)中形成的基可使過氧化氫酶發(fā)生不可WB數(shù)據(jù)處理與圖表制 by阿波沒有第一部分?jǐn)?shù)據(jù)處理（基于ImageJ、 用打酸談補(bǔ)充材料 酸談補(bǔ)充材料 酸談補(bǔ)充材料 第二部分圖表制作（基于Prism、 酸談補(bǔ)充材料 二、WB條帶截?。ɑ赑S用PS打開WB條帶圖酸談補(bǔ)充材料 酸談補(bǔ)充材料 三、組圖（基于PS用PS打開之前輸出的柱狀圖和條帶圖并進(jìn)行合并后輸出為保持格式酸談補(bǔ)充材料 WB寫 by巧巧、梁鸞WesternblotWesternblotCelllysatesweremixedwith1×SDSbuffer.ProteinswereseparatedbySDS-PAGEandthentransferredontoPDVFmembranes.Afterthemembraneshadbeenprobedwithindividualantibodies,theantigen-antibodycomplexwasvisualizedbySuperSignal-enhancedchemiluminescencereagents(Millipore,USA).TheantibodyagainstLRP16(1:500dilution)wasagoatpolyclonalantibodyfromSantaCruzBiotechnology,Inc.AntibodiesofCTNNBIP1,CCND2,DKK4,TCF7,CTBP1,DKK1,FOSL1,FRAT1,andFZD5werefromCellSignalingLRP16preventshepatocellularcarcinomaprogressionthroughregulationofWnt/β-cateninsignaling WesternblottingWesternblottingwascarriedoutusingstandardtechniques.Briefly,cellswerewashedtwicewithPBSandthenlysedwithice-coldlysisbuffer.Theproteinlysatewascollectedaftercentrifugation.TheproteinconcentrationwasfiedusingaBCAassaykit(Pierce,ThermoFisherScientific,Waltham,MA,USA)accordingtothemanufacturer’sinstruction.Equaltyofproteinlysatefromeachsamplewasyzedby10%SDS,followedbyproteintransfertoPVDFmembrane(Millipore).ThemembranewasthenincubatedwithprintibodiesagainstAtg-7,Atg-5,andLC3B-IIautophagyrelated酸談補(bǔ)充材料 genes(AutophagyAntibodySamplerKit#4445,CellSignalingTechnology,Danvers,MA,USA)overnightat4℃,washed,andincubatedwithappropriateperoxidaseconjugatedsecondaryantibodies.ProteinexpressionwasdetectedusingtheusingUVPimagingsystem(UVP,LLC,Upland,CA,USA).Antibodyagainstβ-actinwasusedasaloadingcontrol.4-AcetylantroquinonolBsuppressesautophagicfluxandimprovescisplatinsensitivityinhighlyaggressiveepithelialcancerthroughthePI3K/Akt/mTOR/p70S6KsignalingpathwayMingcheLiuetal.ToxicologyandAppliedPharmacology:ProteinextractionandwesternblotAfterdesignatedtreatment,cellswerewashedtwicewithice-coldPBS,andthetotalproteinconcentrationwasyzedusingthebicinchoninicacidassay(BCA;BeyotimeInstituteofBiotechnology,Beijing,China)accordingtothemanufacturer’sinstructions.Totalcellextracts(50μgtotalprotein)wereresolvedbysodiumdodecylsulfate(SDS)–10%polyacrylamidegelelectrophoresisandtransferredontopolyvinylidenedifluoride(PVDF)membranes.Nonspecificbindingwasinhibitedbyincubatingthemembraneswith8%skimmedmilkinTris-bufferedsaline(TBS)with0.5%Tween-20.Subsequently,membraneswereincubatedwithantibodiesagainstSIRT1,FOXO3a,cleaved-caspase3(Cl.CASP3),cleaved-polyADP-ribosepolymerase1(Cl.PARP1),cytochromec,P53(allfromCellSignalingTechnology,Danvers,MA,USA),p16,γ-H2A.X(bothfromAbcam,Cambridge,MA,USA),p21(SantaCruz,CA,USA),andβ-actin(ZhongshanGoldenBridgeBiotechnology,Beijing,China)overnightat4℃atanappropriatedilution(1:1000).ThemembraneswerewashedwithTBSwithTween-20(TBS-T)and酸談補(bǔ)充材料 thenincubatedwithperoxidase-conjugatedAffinipuregoatanti-rabbitIgG(H+L)andanti-mouseIgG(H+L)-labeledsecondaryantibodies(ZhongshanGoldenBridgeBiotechnology,Beijing,China)dilutedat1:5000for1hour37℃.SpecificcomplexeswerevisualizedonanX-rayfilmElectroChemi-Luminescence(ECL)detectionwithBeyoECLPlus(BeyotimeInstituteofBiotechnology,Beijing,China)followingthemanufacturer’sprotocol.Alldatawereobtainedintriplicate,independentexperiments.RolesofmicroRNA-34aingSIRT1inmesenchymalstemZhangetal.StemCellResearch&Therapy(2015)6:19510.1186/s13287-WesternblotAtofreperfusion,ventricleswerefrozenandreducedtopowderusingliquidN2.Thefrozentissuepowderwashomogenizedincoldbuffercontaining(inmM):20MOPS-TrispH7.0,300sucrose,2EDTA,2EGTAandthedetergents1%NonidetP-40,1%SDSpH7.4,withproteaseinhibitors(2μg/mLleupeptin,1μg/mLpepstatinand1mMbenzamidine[Calbiochem,LaJolla,CA, inhibitor(PhosSTOPTM, Sampleswerehomogenizedusingaglasstissuegrinderandcentrifugedat1000×gfor20minat4℃.Aliquotswerestoredat-80℃forWesternblotysis.Proteinconcentrationwasdeterminedbybicinchoninicacid(BCA)assay.ProteinswereseparatedbySDS(sodiumdodecylsulfatepolyacrylamidegelelectrophoresis)(8%gels)andtransferredontoPVDF(polyvinylidenedifluoride)membranes(MilliporeCorp.,Bedford,MA,USA).Membraneswereprobedwiththefollowingprimaryantibodies:anti-pAkt(S473),anti-Akttotalandanti-β-tubulin.Afterincubationwiththeappropriatesecondaryantibody,antigen–antibodyreactionwasdetectedusingECL酸談補(bǔ)充材料 (Amersham,Biosciences).BlotswerefiedbydensitometricysisusingthesoftwareUN-SCAN-ITgelSilkScientific,Inc.(Orem,UT,USA)andtheresultswerenormalizedwithrespecttoβ-tubulin,aproteinthatdidnotchangeinthisI/Rmodel.Protectionofthemyocardiumagainstischemia/reperfusioninjurybyangiotensin-(1–9)throughanAT2RandAkt-dependentmechanismEvelynMendoza-Torresetal.Pharmacological二、文獻(xiàn)中WesternBlotting圖例及描述c,dWesternblot ysisshoweddose-dependentregulationofSIRT1bymiR-34aaftertransfectionwithmiR-34amimic,NCmimic,miR-34ainhibitor,orNCinhibitorfor72hours,respectively.EachcolumnrepresentsmeanSDfromthreeindependentexperiments.*P<0.05vs.control,△P<0.05vs.transfectionwith10nMmiR-34amimic.miRNAmicroRNA,NCnegativecontrol,ORFopenreadingframe,SIRT1silentinformationregulator1,UTRuntranslatedregionRolesofmicroRNA-34aingSIRT1inmesenchymalstemcells.Zhangetal.StemCellResearch&Therapy(2015)6:19510.1186/s13287-a.EnhancedLRP16expressionwasdetectedbyqRT-PCRandWesternblotLRP16preventshepatocellularcarcinomaprogressionthroughregulationofWnt/β-cateninsignaling 第一句（Westernblotting是按照標(biāo)準(zhǔn)方法進(jìn)行的Westernblottingwasconductedunderstandardprocedures/performedaccordingtostandardmethodsaspreviouslydescribed.Western ysiswasperformedusingx/usingantibodiesagainst（使用的抗體也可以在稍后描述第二句（蛋白獲取XcellswerelysedtoobtainproteinsusingProteinsampleswerepreparedwithRIPAlysisProteinsfromxwereextractedfollowingstandardprotocolsaccordingtothemanufacturer’sprotocol.第三句（大致實(shí)驗(yàn)步驟Proteinswereseparatedby8%SDS–PAGEandthentransferredtoPVDFmembrane(Bio-Rad,Hercules,CA,USA).Afterblockingin5%nonfatmilk,themembraneswereincubatedwiththefollowingprimaryantibodies:x;thenthefollowingsecondaryantibodieswereused:x.ProteinswereloadedontoSDS(10–15%)gels,separatedelectrophoretically,andtransferredtoPVDFmembranes(Millipore,USA).Afterblockingwith5%driedskimmilkfor3hatroomtemperature,themembraneswereincubatedovernightat4°Cwithpri Afterseparationbysodiumdodecylsulfatepolyacrylamidegel,proteins(30μg)weretransferredtoaPVDFmembrane(Millipore,Boston,MA,USA).Afterblockingwith5%nonfatmilk,thePVDFmembranewasincubatedwithpri ntibodyinacoldroomovernight.AfterthreewasheswithTris-bufferedsalineTween(TBST)for15min,thePVDFmembranewasincubatedwithperoxidaseconjugatedsecondaryantibody.第四句（條帶可視化TheimmunoreactiveproteinbandswerevisualizedbyECLTheproteinwasvisualizedwithECLdetectionTheblotsonthemembraneweredevelopedwith第五句（獲取TheimageswereacquiredusingaBio-SpectrumGelImagingSystem(UVP,USA).TheresultswererecordedbyphotographingwithaKODAKImageStation(4000Pro,KODAK,Shanghai,China).第六句（數(shù)據(jù)處理TheintensityoftheproteinbandswasfiedwithImageJandnormalizedtothatofGAPDH.TheintegrateddensityoftheblotswasdeterminedbythesoftwareImageJ,andpresentedasapercentageoftheinternalcontrolβ-actin.Westernblottingwasperformedaccordingtostandardmethodsaspreviouslydescribed[x]usingantibodiesagainstx.α-Tubulin(Sigma,SaintLouis,MO,USA)wasdetectedasaloadingcontrol.Westernblotwasperformedaspreviouslydescribed[x].SeeAdditionalfilexfordetailsandantibodiesused.Westernblotysiswasperformedusingx.Tocontrolsampleloading,theblottingmembraneswerestrippedandre-probedwithananti–α-tubulinantibody(1:5000,Sigma,SaintLouis,MO,USA).NuclearextractswerepreparedusingtheNuclearExtractionKit(ActiveMotif),accordingtothemanufacturer’sinstructions.Westernblotwasconductedunderstandardprocedures[x].Briefly,cellswerelysedtoobtainproteinsusingRIPA.Proteinswereseparatedby8%SDS–PAGEandthentransferredtoPVDFmembrane(Bio-Rad,Hercules,CA,USA).Afterblockingin5%nonfatmilk,themembraneswereincubatedwiththefollowingprimaryantibodies:x;thenthefollowingsecondaryantibodieswereused:x.Allantibodiesweredilutedinnonfatdrymilk.TheimmunoreactiveproteinbandswerevisualizedbyECLKit(Pierce,ThermoFisherScientific,IL,USA).TheexperimentwasperformedthreeseparateThetotal,mitochondrial,andcytoplasmicproteinsfromxcellsandtumourtissueswereextractedfollowingstandardprotocolsaccordingtothemanufacturer’sprotocol,andtheproteinconcentrationwasdeterminedusingaBCAproteinassaykit.ProteinswereloadedontoSDS(10–15%)gels,separatedelectrophoretically,andtransferredtoPVDFmembranes(Millipore,USA).Afterblockingwith5%driedskimmilkfor3hatroomtemperature,themembraneswereincubatedovernightat4°Cwith ntibodies(listedinSupportingInformationTable).Afterincubationwithhorseradishperoxidase-conjugatedantibodiesfor2hatroomtemperature,anenhancedchemiluminescencemethodwasusedfordetection,andimageswereacquiredusingaBio-SpectrumGelImagingSystem(UVP,USA).BandswerenormalizedwithGAPDHasaninternalWesternblotwasconductedaspreviouslydescribed[x].Briefly,proteinsampleswerepreparedwithRIPAlysisbuffer(Sigma-Aldrich,Cambridge,MA,USA)withproteaseandphosphataseinhibitorcocktails(ThermoFisherScientific).Afterseparationbysodiumdodecylsulfatepolyacrylamidegel,proteins(30μg)weretransferredtoaPVDFmembrane(Millipore,Boston,MA,USA).Afterblockingwith5%nonfatmilk,thePVDFmembranewasincubatedwithpri ntibodyinacoldroomovernight.AfterthreewasheswithTris-bufferedsalineTween(TBST)for15min,thePVDFmembranewasincubatedwithperoxidaseconjugatedsecondaryantibody.ThenafterthreewasheswithTBSTfor15min,theproteinwasvisualizedwithECLdetectionsolution(Millipore).Thepri werelistedasfollows:x.GAPDHwasusedasinternalcontrol.TheintensityoftheproteinbandswasfiedwithImageJandnormalizedtothatofThetotalproteinswereextractedfromthecells;theproteinlevels酸談補(bǔ)充材料 fiedbytheBio-Radproteinassay.SDSwasperformedtofractiontheproteins,whichweretransferredontoaPVDFmembrane.Themembranewasblockedwith5%skimmilkfor30minatroomtemperature,incubatedwiththeprintibodies(0.5μg/ml)overnightat4°C,andfollowedbyincubationwiththesecondaryantibodies(conjugatedwithperoxidase)for1hatroomtemperature.TheblotsonthemembraneweredevelopedwithECL.TheresultswererecordedbyphotographingwithaKODAKImageStation(4000Pro,KODAK,Shanghai,China).TheintegrateddensityoftheblotswasdeterminedbythesoftwareImageJ,andpresentedasapercentageoftheinternalcontrolβ-actin.ForWBpartwriting,itincludestwoparts:Methods(Reagents,antibodies,animals,tissuecollectionandWB)&Results(showingtheWBresults:representativeblotsandfigures)InBlackisthemanuscript;InBlueistheReferncePaper:LiuSJ*,ZhengP*,WrightD,etal.Sodiumselenateretardsepileptogenesisinacquiredepilepsymodelsreversingchangesinproteinphosphatase2Aandhyperphosphorylatedtau.Brain.139(Pt7):1919-1938,MaterialandReagentsandantibodies.Antibodies&Companyname,forUS,specifictotheStateName,PlustheECLcompany(Areagentalso)TherabbitpolyclonalantibodiespS198andpS262,whichrespectivelyrecognizedphospho-tauatSer198and262,werepurchasedfromEpitomics(Burlingame,CA).ThemousemonoclonalantibodyTau-5,whichrecognizedtotaltau,waspurchasedfromBDBiosciences(SanJose,CA).酸談補(bǔ)充材料 Themousemonoclonalanti-PP2AcandPR55werepurchasedfromMilliporeCorporation(Temecula,CA).Glyceraldehyde-3-phosphatedehydrogenase(GAPDH)wasusedasloadingcontrolandrecognizedbyrabbitmonoclonalanti-GAPDHbody,whichwaspurchasedfromCellSignallingtechnology(Danvers,MA).Bicinchoninicacidproteinassaykit(BCAkit)waspurchasefromPierceBiotechnologyInc.(Rockford,IL).PP2AimmnunoprecipitationphosphataseassaykitwaspurchasedfromMilliporeCorporation(Temecula,CA).EnhancedchemiluminescencedetectionkitwaspurchasedfromGEhealthcareInc.(LittleChalfont,Buckinghamshire,UK).SodiumselenatewaspurchasedfromSigma-AldrichInc.(SaintLouis,MO).Generalchemicals,suchassodiumdeoxycholate(DOC),sodiumdodecylsulphate(SDS)andIGEPALCA-630(NP-40),werepurchasedfromSigma-AldrichInc.(SaintLouis,MO).Osmoticmini-pumpswerepurchasedfromDURECTCorporation(ALZET?models2004and2006,Cupertino,CA).Experimentalanimals.(Animalspecies,housinginfo&EthicsAdultmaleWistarratswereusedinallexperiments.TheseratswereobtainedfromourbreedingcolonyintheDepartmentofMedicine(RMH),UniversityofMelbourne,individuallyhousedandmaintainedon12hlight/darkcycleswithfoodandwateravailableadlibitum.AllanimalexperimentswereapprovedbytheAnimalEthicsCommitteesofTheUniversityofMelbourne,andwereperformedinaccordingwiththeguidelinessetbytheAustralianNHMRCCodeofPracticefortheCareandUseofAnimalsforScientificPurpose.Tissuecollectionandprocessing.(Timepoints,tissuecollectionmethod,braindissectionparts,storingmethod)Twenty-fourhoursafterthelastkindlingstimulation,orafterthefinal-EEGrecordinginthepost-SEmodel,animalsweresacrificedwithalethaldoseofpentobarbital.Thebrainswererapidlyremovedandsplitintotwo3mMKCl,6mMMgCl2,1mMCaCl2,1.25mMNaH2PO4,25NaHCO3,10.6mMglucose.Thelefthemispherewasfixedwith4%paraformaldehydefor48hoursat4°C.Therighthemispherewasmicro-dissectedtoextractthreeregions:amygdala,hippocampusandcortex,whicharehighlyrelevanttokindledseizures.Theblocksoftissuewererapidlyfrozeninliquidnitrogenandat-80°C.Westernblotting.(Homogenizationmethod,proteinextractsmethod:RIPAmostly,proteinficationwithBCAorLowell,Gelmakingprocedureconcentration,%!isimportant,membranestype,transfermethod,blottingantibodies1stand2nd,exposuremethodandloadingcontrol)ThefrozentissuesweregroundondryiceanddissolvedinRIPAbuffer(50mMTris(pH7.4),150mMNaCl,0.1%sodiumdodecylsulphate(SDS),0.5%sodiumdeoxycholateand1%NP-40)withproteaseinhibitorscocktailandphosphataseinhibitorscocktail.Thebrainextractswillbecentrifugedat12,000×gfor15minutesat4°CandthesupernatantwasusedforWesternblotting.AftertheproteinconcentrationofsupernatantwasdeterminedwithBCAkit,thesupernatantwasmixedwithin5:1(v/v)ratiowithsamplebuffercontaining300mMTris-HCl(pH6.8),300mMdithiothreitol,12%SDS,0.6%bromophenolblue,and60%glycerol,boiledfor10minat95°C,thencentrifugedat12,000×gfor10min,andthesupernatantwasstoredat-80°CforWesternblottingysis.WeseparatedthetissueproteinswithSDS-polyacrylamidegelelectrophoresis(SDS),andelectro-blottedthebandsofproteinsontopolyvinyldifluoride(PVDF)membranes.Next,wedevelopedtheblotsonPVDFmembraneswithPS396(1:10,000),PS198(1:2000)andpS262(1:1000),respectively.Then,thesemembraneswerestrippedandreprobedforTau-5(1:1,000)andthenGAPDH.TomeasuretheexpressionofPP2A,werantheotherWesternblotting,whichareseparatedfromtauWesternblotting.WefistblottedthePVDFmembraneswithanti-PR55(1:1,000)andthenanti-PP2Ac(1:1,000)afterstripped.Thesemembraneswerestrippedagainandreblottedbyanti-GAPDH(1:10,000).Allproteinblotswerevisualizedbyenhancedchemiluminescentsubstratekitandexposuretox-fi.TheseblotswerescannedandthemeanintensityoftheblotswasfiedusingNIHImageJsoftware(Abramoffetal.,2004).Theratioofimmunoreactivity(IR)associatedwithphospho-tau(pS198andpS262)tototaltau(Tau-5)andotherproteinstoloadingcontrol(GAPDH)wascalculatedandtheresultswereexpressedasrelativeoftheaveragecontrolvalue(shamoperation,sodiumchloridetreatmentascontrol).Statisticalyses.(ShouldincludesomebriefinforegardingtheWBStatisticalcomparisonswereperformedusingSPSS20.0.Westernblotting,PP2Aactivity,andthenumberanddurationofseizures/daywereysedwithindependent-samplesttests.RepeatedmeasuresANOVAwereusedtoassessseizuredurationandseverit