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class="pro_detail_btn"> <a href="#order" class="prodtl_btn1">產(chǎn)品咨詢</a><a href="/contact.html" target="_blank" class="prodtl_btn2">聯(lián)系我們</a> </div> </div> <div id="sbrjztcv42p" class="clear"></div> </div>  <div class="sbrjztcv42p" id="ny_pro_box02"> <div class="sbrjztcv42p" id="con"> <ul id="tags"> <li id="sbrjztcv42p" class=selectTag><A onmouseover="selectTag('tagContent0',this)" href="javascript:void(0)"onFocus="this.blur()">產(chǎn)品介紹：</A> </li> </ul> <div class="sbrjztcv42p" id=tagContent> <div id="sbrjztcv42p" class="tagContent selectTag" id=tagContent0><p><strong>Western免疫印跡</strong></p><p> Western Blot采用的是聚丙烯酰胺凝膠電泳，被檢測物是蛋白質(zhì)，“探針”是抗體，“顯色”用標(biāo)記的二抗。經(jīng)過PAGE分離的蛋白質(zhì)樣品，轉(zhuǎn)移到固相載體（例如硝酸纖維素薄膜）上，固相載體以非共價鍵形式吸附蛋白質(zhì)，且能保持電泳分離的多肽類型及其生物學(xué)活性不變。以固相載體上的蛋白質(zhì)或多肽作為抗原，與對應(yīng)的抗體起免疫反應(yīng)，再與酶或同位素標(biāo)記的第二抗體起反應(yīng)，經(jīng)過底物顯色或放射自顯影以檢測電泳分離的特異性目的基因表達的蛋白成分。該技術(shù)也廣泛應(yīng)用于檢測蛋白水平的表達。</p><p><strong>Western Blot操作步驟</strong><br />（一）蛋白質(zhì)樣品獲得：細(xì)菌誘導(dǎo)表達后，可通過電泳上樣緩沖液直接裂解細(xì)胞，真核細(xì)胞加勻漿緩沖液，機械或超聲波室溫勻漿0.5-1min。然后4℃，13,000g離心15min。取上清液作為樣品。<br />（二）電泳：制備電泳凝膠，進行SDS-PAGE。<br />（三）轉(zhuǎn)移：（半干式轉(zhuǎn)移）<br />1、電泳結(jié)束后將膠條割至合適大小，用轉(zhuǎn)膜緩沖液平衡，5min×3次。<br />2、膜處理：預(yù)先裁好與膠條同樣大小的濾紙和NC膜，浸入轉(zhuǎn)膜緩沖液中10min。<br />3、轉(zhuǎn)膜：轉(zhuǎn)膜裝置從下至上依次按陽極碳板、24層濾紙、NC膜、凝膠、24層濾紙、陰極碳板的順序放好，濾紙、凝膠、NC膜精確對齊，每一步去除氣泡，上壓500g重物，將碳板上多余的液體吸干。接通電源,恒流1mA/cm2，轉(zhuǎn)移1.5hr。轉(zhuǎn)移結(jié)束后，斷開電源將膜取出,割取待測膜條做免疫印跡。將有蛋白標(biāo)準(zhǔn)的條帶染色，放入膜染色液中50s后，在50%甲醇中多次脫色，至背景清晰，然后用雙蒸水洗，風(fēng)干夾于兩層濾紙中保存，留與顯色結(jié)果作對比。<br />（四）免疫反應(yīng)：<br />1、用0.01M PBS洗膜，5min ×3次。<br />2、加入包被液，平穩(wěn)搖動，室溫2hr。<br />3、棄包被液，用0.01M PBS洗膜，5min×3次。<br />4、加入一抗（按合適稀釋比例用0.01M PBS稀釋，液體必須覆蓋膜的全部），4℃ 放置12hr以上。陰性對照，以1%BSA取代一抗，其余步驟與實驗組相同。<br />5、棄一抗和1%BSA，用0.01M PBS分別洗膜，5min×4次。<br />6、加入辣根過氧化物酶偶聯(lián)的二抗（按合適稀釋比例用0.01M PBS稀釋），平穩(wěn)搖動，室溫2hr。<br />7、棄二抗，用0.01M PBS洗膜，5min×4次。<br />8、加入顯色液，避光顯色至出現(xiàn)條帶時放入雙蒸水中終止反應(yīng)。</p><p><strong>Western Blot注意事項</strong><br 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