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Western Blot 中你不可不会的剥离技术

发布: 2015-11-16 983阅读 209评论


Western Blot的过程复杂繁琐，大家难免会在实验中遇到一些问题。而且整个实验耗时较长，所以我们要尽可能的利用现有的转渍膜和蛋白样品。如此一来，剥离和再探测的技术也就应运而生。

 

为什么要剥离？

有科学依据的原因：

  1. 蛋白质样品或是昂贵或是有限的

  2. 用相同的或是不同的抗体来重新确认实验结果；

  3. 蛋白样品可以使用不同的抗体来分析，这样在比较相对含量的时候非常有用。例如，可以测量磷酸化的蛋白质在总蛋白中的含量。

在实验室中更“接地气”的原因：

  1. 去除用错了的抗体（不要不相信，真的有小伙伴加错过）或是使用了不合适的抗体浓度；

  2. 省去重新跑胶和转渍的时间；

  3. 去除掉不合适的封闭液，重新来过。

 

怎么剥离？

了解了原因之后，大家是不是觉得多多少少都想尝试着在Western Blot之后进行一下剥离再探测呢。那么现在，小编就给大家讲讲三种不同的剥离技术。每种技术的目的都是尽可能的去除掉结合的抗体并且尽最大可能保证转渍膜上蛋白质样品的数量和质量。

方法一：低pH值甘氨酸溶液

这种方法通常被称为“温和剥离法”。这种方法操作起来很简单，使用低pH值的甘氨酸溶液让结合的抗体离解。低pH值之所以能离解结合的抗体，是因为它可以使抗体-抗原的结合位点失活。这种溶液的配方是将固体的甘氨酸溶进水中，并且用浓酸把pH 调整到2.2。通常情况下还要加入0.1–1% SDS (w/v) 来增加蛋白质的离解。转渍膜在抗体剥离缓冲液里的培养时间是30-60分钟。值得注意的是，如果大家要二次利用抗体，千万不要在缓冲液里加SDS。SDS可以使蛋白质失活，抗体也不例外啦！而且要记得重新调整pH再使用回收的抗体，否则抗体很可能还没有回复原本的结构！

方法二：加热和洗涤

这个方法就没有那么温和了，但是对信号强的Western Blot很适合。这种方法可以改变抗体的二次结构，使其脱离原本结合的抗原（蛋白质样品）。在使用前，通常在中性的Tris-HCl溶液加入还原剂，例如2-巯基乙醇和SDS。在转渍膜中加入适量的调制好的缓冲液，再将缓冲液加热到50-80度，持续45分钟即可。值得注意的是，这个方法加入了还原剂，所以回收之后的抗体已经失活并且是活性是不可逆的，就不能再次使用了。与此同时，清洗转渍膜一定要彻底，否则新加的抗体也可能在残余的还原剂中失活。

方法三：商品化抗体剥离缓冲液

这些抗体剥离缓冲液都是有专利的，所以我们对其中的配方不得而知。但是商家的介绍大都表示比以上两种方法更温和。因为不需要加入硫醇，更不需要高温。神奇的是，在需要的时间比实验室里的自制办法要短的情况下（有的只需要15分钟），剥离效率几乎没有被影响。小编在这里给大家举几个例子，Millipore的ReBlot，Pierce的 Restore。大家还可以在网上搜到更多，通过比较，一定能找到适合自己实验的抗体剥离缓冲液的！

11031
剥离技术的介绍就结束啦，希望对大家有用。在接下来的文章中，小编会跟大家说说在剥离之前的一些注意事项，敬请期待！


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