膜再生液(抗体去除液、抗体剥离液) 温和型 P1650空前钜惠,买三送一,500ml,时间:2024年11月1日-12月31日!
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描述:
膜再生液,也称做抗体剥离液体、一抗二抗去除液。允许对同一张膜进行多次Western Blot检测。在室温条件下,将用过的膜浸泡在膜再生液中30分钟,能够选择性清除与膜抗原结合的第一抗体及第二抗体,但不影响电转移到膜上的蛋白。随后可以使用不同的抗体进行下一轮Western Blot实验,可利用同一张膜进行多次蛋白检测。不仅适宜用少量样品多次检测多种不同蛋白,即使样品量并不匮乏,采用这种多次检测同一张膜上的蛋白的策略,能省去给药处理、蛋白电泳和膜转移等耗费性步骤。
特点:
(1)无毒无味无害,室温保存和使用;
(2)可对同一张膜进行10次以上的Western Blot检测。
适用:
硝酸纤维素膜或PVDF膜。使用前膜可以保存在PBS或TBS缓冲液,也可以室温干燥保存数月。
干的PVDF膜应该先用甲醇泡5分钟。
安全性:
无毒无味。按照普通化学品安全规范进行操作和处置。
储存:
室温保存,一年有效。温度过低可能出现浑浊。
效果展示:
第一次Western Blot 第一次Western Blot后用膜再生液处理,然后进行第二次Western Blot
操作步骤:
1. 将膜充分浸泡于适当体积的再生液中,室温孵育15~30分钟并不时晃动。孵育时间的长短应参考后面的说明进行优化。洗脱某些抗体需要较长的时间如30~60分钟。
2. 用镊子取出膜,用自备Western Blot洗涤缓冲液或普利莱封闭洗涤缓冲液(B1009)淋洗膜一次,再洗膜5分钟。
3. 此时膜上抗体已去除,膜已再生。用脱脂奶粉或BSA封闭,进行下一轮Western Blot实验。
说明:
1. 膜再生或Stripping的实质是在不影响膜上结合的抗原的条件下,将与抗原分子结合的一抗和二抗洗脱下来。有许多因素影响抗体从膜上的洗脱,如膜的类型、抗体类型和浓度及其与抗原结合特性等。按下面的说明优化再生液中孵育膜的时间至关重要。
2. 确定膜上抗体是否去除与优化再生液中孵育膜的时间:用ECL工作液孵育再生后的膜约1分钟,然后进行X光胶片曝光并显影,可确定膜上的抗体是否完全去除。如显示条带,表明抗体未完全去除,应继续将膜浸泡在再生液中孵育30~60分钟。然后再次ECL 检查膜上抗体是否去除。重复此步骤直到抗体完全去除并确定最佳孵育时间。
3. 由于至今尚不清楚的原因,使用脱脂奶粉封闭的膜要比使用BSA封闭的膜上的抗体更容易被Strip下来。因此准备进行Strip的膜,应该使用脱脂奶粉而不是BSA封闭。
4. 尽量避免使用干燥保存的膜,因为干的膜上的抗体很难被Strip干净。
部分使用普利莱膜再生液发表的SCI文章,供参考:
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