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蛋白提取及WB
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===WB=== [[File:WB实验.jpg|center|thumb|300px|WB实验]] <ol> <li>剪裁目标蛋白和参比蛋白的膜,浸入封闭液中,摇床15min(按封闭剂的种类) <li>TBST配制: <table class="wikitable" style="margin: auto; border-collapse: collapse; border: 1px solid #a2a9b1;"> <tr> <th colspan="2">0.5L TBST缓冲液组成</th> </tr> <tr> <th>Reagent</th> <th>Quantity (for 0.5 L)</th> </tr> <tr style="border-top: 1px solid #a2a9b1;"> <td>Tris</td> <td>1.21 g</td> </tr> <tr> <td>NaCl</td> <td>4 g</td> </tr> <tr> <td>UP水*</td> <td>0.5 L</td> </tr> <tr> <td>吐温20**</td> <td>2.5 mL(0.5%)</td> </tr> <tr style="border-top: 1px solid #a2a9b1;"> <td colspan="2"><i>*:Tris和NaCl溶解后HCl调整pH至7.6(pH计)</i></td></tr> <tr style="border-top: 1px solid #a2a9b1;"> <td colspan="2"><i>**:调完pH再加吐温,使用用特殊的枪和吸头</i></td></tr> </tr> </table> <li>回收封闭液,TBST清洗5次(摇床5 min) <li>加一抗5ml(用3%脱脂奶粉in TBST按照说明书稀释),膜浸透,避免漂浮或粘壁,37度摇床1h,4℃过夜 <li>回收一抗,膜用TBST清洗5次,每次摇床5min后弃去,去除残余一抗 <li>加入二抗(用3%脱脂奶粉稀释),37度摇床1h,TBST清洗5次,最后将膜保存在TBST中 <li>使用314的BioRad凝胶成像系统,image lab软件,将缓冲液中保存的膜浸泡至显影液(按说明书配制)中2min,转移至培养皿,放入样品盘,成像 <li>仪器: <ol> <li>开机:插排-稳压电源-仪器-电脑(关机反方向) <li>image lab软件:运行软件-确定-select scanner-chom...-select-chem Hi sentilye(取消VVA勾选) [[File:WB结果例图.png|center|thumb|300px|WB结果例图]]
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