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蛋白提取及WB
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===制胶=== <ol type="1"> <li>支架下方垫橡胶垫实现密封,薄板朝外装载玻璃板,对准刻度(吸头辅助夹紧)[[File:SDS-PAGE制胶装置.jpg|center|thumb|300px|SDS-PAGE制胶装置]] <li>配制电泳液:每次用量约1.5 L <table class="wikitable" style="margin: auto; border-collapse: collapse; border: 1px solid #a2a9b1;"> <tr> <th colspan="2">1.5L SDS-PAGE电泳液组成</td> </tr> <tr> <th>Reagent</td> <th>Quantity (for 1.5 L)</td> <tr> <td> Tris</td> <td>4.545 g</td> <tr> <td> 甘氨酸</td> <td>21.6 g</td> <tr> <td>SDS</td> <td>1.5 g</td> <tr> <td> UP水</td> <td>1.5 L </tr> </table> <li>根据目标蛋白的分子量,按照商品化制胶试剂说明书调配试剂,制作分离胶和浓缩胶,倒入玻璃板,分离胶灌至距绿边1cm,浓缩胶灌满,插入梳子(刻度向外),注意观察梳齿气泡。[[File:SDS-PAGE凝胶比例.jpg|center|thumb|300px|SDS-PAGE凝胶比例]] <li>凝固后,将玻璃板转移至电泳槽中,薄板向里,薄板上沿对齐标线,下沿不用触底,向夹层中倒入电泳液,验漏(是否夹紧,否则电泳时功率不稳) <li>拔出梳子,用加样吸头吸取电泳液清洗样品孔,排气泡以及排除残余胶液 <li>上样及marker,一般1孔20ug,最多30ul,10孔梳10-15 ul/孔,15孔梳7-10 ul/孔;最侧边的孔及空余孔上样 1xloading buffer(电泳液稀释) <li>电泳电压:浓缩胶约90V(W约为4W),分离胶约150V(W约为8W),室温 <li>溴酚蓝迁移至凝胶2/3处即可停止电泳[[File:SDS-PAGE电泳例图.jpg|center|thumb|300px|SDS-PAGE电泳例图]] </ol>
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