相信很多同学还在纠结如何优化蛋白质印迹法(Western Blot, WB)的操作方法(protocol),那么其中一个需要注意的细节就在于转渍(transfer)前要平衡转渍膜(equilibrate the membrane)。但不少人会有所疑虑,这看起来无关紧要的一步,到底需不需要做呢?小编之前也有相同的疑虑,做了很多回WB,都没有提前平衡转渍膜。但是后来发现这小小一步竟然是有很大影响的。今儿小编就给大家简单讲讲什么是平衡膜和三个需要它的原因。
在WB中,平衡膜这个步骤指的是用转渍缓冲液(transfer buffer)浸泡转渍膜和/或凝胶至少五分钟。要注意的是,这个步骤和最开始的膜激活(activation of membrane)是不同而且不冲突的。平衡膜是在跑完电泳(gel electrophoresis)后和转渍前的一步。
看上去非常简单,那么它的作用是什么呢?
1它能除掉电泳缓冲液中的污染盐 (contaminating electrophoresis buffer salts)
这些盐会增加转渍缓冲液的导电性(conductivity),从而增加了整个转渍系统的热量产生。如果你用PVDF膜的话就更需要小心了,因为它对十二烷基硫酸钠(SDS)的浓度很敏感。SDS浓度太高的话会抑制蛋白的结合能力,甚至让已经结合的蛋白脱落。
2它可以让你的凝胶在转渍前收缩到不会变的大小(shrink to its final size)
这个需要考虑两个方面:其一,转渍系统中产生的热量会让凝胶膨胀扩大;其二,聚丙烯酰胺凝胶(polyacrylamide gel)通常会在含有甲醇的溶液里收缩(大部分转渍液都用甲醇!)。因此,可以提前冷藏转渍液并且把凝胶浸泡在转渍液里至少五分钟。但是如果你的目标蛋白很小的话,要确保平衡凝胶少于五分钟,否则分子量较小的蛋白就会从凝胶中扩散(diffuse)到溶液中。
3它可以防止凝胶和转渍膜变干
如果你正好有别的实验要忙,那不妨把凝胶和转渍膜浸泡在转渍液里等待一下,这样可以很好的安排别的时间。根据网上经验,使用PVDF膜和半干转渍系统转移高分子量的蛋白,浸泡两个小时是不影响转渍效率的。但注意这只适用于分子量比较大的目标蛋白。而且如果你有所怀疑的话,还是不要等太久。
所以到底要不要平衡呢?其实还是取决于你的转渍系统:
湿转渍(Wet transfer): 需要!
半干转渍(Semi-dry transfer): 需要!
快速真空转渍(Rapid vacuum transfer): 不需要! 这样的系统不需要电流回路,所以污染盐不会影响。而且转渍很快,凝胶也不会收缩。
图片来自网络,内容由BioEngX原创,转载请注明!
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