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Western Blot经验谈

作者: Viktor. Z 发布: 2016-05-17 11,634阅读 361评论

本文为BioEngX-Western blot 科研讨论群第一次Study club的演讲报告，演讲人为来自英国Newcastle 大学的Victor学士

大家好，首先感谢BioEngX组织这次活动（Study Club），也很荣幸能获得这次讲座的机会。今晚我会讲一下，Western Blot在我的研究课题中的作用，以及分享一些自己操作Western Blot的经验。希望对大家有帮助。

我叫Victor，目前在英国纽卡斯尔大学细胞与分子研究院（ICaMB）就读博士三年级。我2013年本科毕业于纽卡斯尔大学，主攻生物化学与免疫学，随后加入了纽卡斯尔大学下属的细胞与分子研究院－核糖体生物学研究组（Ribosome Biology Group）开始了博士学业。

课题简介

我博士的课题主要集中在核糖体合成、核糖体亚基蛋白(ribosomal subunit)、核糖蛋白-rRNA复合体(RNP) 与肿瘤蛋白p53之间的信号通路关系，同时也关注核糖体蛋白磷酸化与蛋白功能对信号通路的影响。

当细胞受到来自外环境不同的应激压力（stress，如UV造成的DNA损伤，缺氧应激oxidative stress，失控表达的原癌基因misregulated oncogene expression等） p53蛋白的表达负责调控 细胞周期停顿(cell cycle arrest)、DNA修复、细胞凋亡(apoptosis)等等应对各种应激压力细胞生理反应。

核糖体的合成会消耗细胞的大部分能量，但同时核糖体对细胞的生存至关重要。核糖体合成过程出错会导致p53蛋白激活。5S RNP通过抑制HDM2蛋白对p53的抑制功能，从而导致p53蛋白表达水平的提升。研究也显示核糖体合成出错与部分罕见疾病有关联（如Diamond Black fan Anemia, Treacher Collins Syndrome, 5q-syndrome 等等）其病人随后患急性髓性白血病AML等疾病的机会亦有所提升。因此通过这个研究我们能进一步了解p53的功能如何通过核糖体亚基被调控，这将对癌症以及其他疾病的治疗非常有益。

Western blot在我研究中的作用

Western blot是一种简单却非常高效的蛋白质检测技术，具有高特异性高灵敏度，半量化和周期相对短等等优点。更由于SDS-PAGE具有高灵活性，高兼容性的特点，在施行各种研究主体为蛋白质的分子生物学实验之后，用Western blot检测蛋白质有否存在、存在量多少，十分有用。

在我的研究中，Western Blot主要用于以下的实验：重组蛋白质纯化后验证蛋白量、体外蛋白－蛋白之间关联度实验（in vitro protein-protein interaction analysis）后检测目标蛋白是否关联、RNA干扰效果的确定（RNA interference efficiency confirmation）、细胞信号通路研究以及蛋白磷酸化。下面我简单进行介绍。

1用于in vitro 试验的重组蛋白质表达和纯化

这点是与蛋白蛋白相互作用研究密切相关的。简单来说就是，我们会为重组蛋白加上一个标签，比如经典的His/FLAG/HA tag之类的。然后我们利用E. coli对蛋白质进行表达，而后应用各种技术对蛋白质进行提纯。提纯后的蛋白就可以用于进行体外的蛋白－蛋白相互作用的研究。Western blot 用于检测蛋白表达量、提纯量、是否存在特异性关联等等

2RNA干扰效果的确定

在人类细胞组织培养中，我们会用siRNA将某些特定蛋白knock  down（在mRNA层面减少蛋白质表达量）。因为我们的目的是减少蛋白的表达量，最后会用western blotting的方法验证蛋白质的表达量。

3细胞信号通路研究

Western blot 用来检测p53蛋白活性及表达量情况。在研究中，我们会利用一些药物处理细胞或利用基因工程手段敲除某些基因以后，查看p53的表达量。为了衡量p53活性，我们更会对p53下游某些基因，例如p21的表达量进行检测。

4蛋白磷酸化

Western blot之前要进行SDS-PAGE。有一种技术叫作Phos-tag，在SDS-PAGE的凝胶中引入一种特别的化学物，就能够减慢磷酸化蛋白的迁移速度，达到分离磷酸化后蛋白的目的。Western blot 用于检测磷酸化前后蛋白的分离度。

Western blot 经验谈

接下来跟大家分享一下我积累的操作western blot的小技巧。

1跑SDS-PAGE的时候，选择正确的凝胶浓度十分重要

可以看下面的例子：

1      图1

图1是已经转过膜的两张SDS-PAGE。左边是10%的凝胶，右边是13%的凝胶，两张胶在同一个电泳槽内跑了相同的时间。两张胶的蛋白质ladder也是一样的，最小的一条带（最下边）的大小是15 KD，红色的带的大小70 KD，红色带上面的一条带的大小是100 KD。左右两张胶的相同之处在于，100-70 KD区间内蛋白的分离度是相近的。两张胶的不同之处在于对于小体积蛋白质的分离度，从图中可见下面的条带尤其是35-15 KD之间的蛋白的分离度非常不一样。通过这个简单的对照可知，凝胶浓度对于实验的影响是很大的。

2上样量（loading）和凝胶对蛋白分离度的上限

上样量的多少对western blot的清晰度有很大的影响，我们知道SDS-PAGE对于可分离的蛋白质浓度是有上限的，如果上样量过多会怎么样呢？我们先来看一张应用了正确的上样量的膜是怎样一种情况。

2

图2 正常上样量

先看一张正常上样量的膜。图2是一张转好了的膜，中间是预染的marker，红色的条带是蛋白质。将蛋白质染成红色，应用了ponceau S。这种蛋白染料的最大好处是染色脱色都非常快，而且不会对随后的抗体造成影响，因此十分适合用于转膜后确认转膜质量（有没气泡）。从图中可知，不同分子质量的蛋白质之间分离度非常好，同时泳道与泳道之间分离的效果也很好。

我们现在来看一张上样量过多的凝胶：

Picture1

图3 上样量过多

现在再看一个过度上样的例子。从图3中可见，左边第一条泳道是一片模糊的东西，蛋白质没有清晰的被分离开，这显示这次实验的上样量过高，超过了这张胶所能分离蛋白的浓度上限。如果拿这样的胶进行下一步的western blot，与一抗，二抗（primary / secondary antibody）进行孵育，结果也不会看到清晰的条带，会看到模糊一片的信号。

3充分利用每一张胶

根据实际情况，跑胶时我有时候会将胶切开，一半的胶用来进行考马斯亮蓝染色，另一半的胶用来转膜进行western blot。转完膜后，不同区间的胶可以用来去做不同的抗体。 具体见下图。

4

图4

图4是我刚刚跑好的SDS-PAGE, 从图中可见两条预染的protein marker。一张胶其实一条Marker就足够了。为什么我要load两条Marker呢？看了下面的图你就会知道了。

 

5

图5

从图5中可以看出，我是从左边的Marker中间将胶切开了，这样做的目的是用切下去的左边那块胶进行考马斯亮蓝染色，查看蛋白质表达情况，剩余的这两块胶上的样品与切掉的左边胶上的样品是一样的，用来进行western blot 特应性的检测某一些蛋白。6

图6

从图6可以看到，一张胶被分成了三块，右边的是考马斯亮蓝染色的结果，左边的两张膜目前处于封闭（blocking）的阶段，封闭液用的是奶粉，后面会用来抗不同的抗体。通过这种方式，不但能够提高实验的效率，还能够方便后期的比较，因为蛋白是跑在同一张胶上的，所以能够没有误差的进行对比。

4将两张mini胶拼在一起，一次性转膜

应用这个小技巧时，两张胶要同一轮跑开（切记每一张mini胶上都要有marker）。待两张胶同时跑完胶后，转膜前可以依据marker把两张胶拼接起来，这样可以保证对样品操作的一致性，减少误差。

7

图7

同样的想法，我将一张胶切成了四份（图7）。其中一份我用来做了内参，下面几个部分用来抗不同的蛋白，这就相当于跑一次胶，转一次膜却做了好几次实验。

5选择抗体

应用了上面的技巧后，如果你想再进一步提升实验的效率，你可以用不同的抗体。比如说，你把两个不同的抗体，一个来自老鼠，另一个来自兔子，你将两个抗体放在一起进行实验，你一次就能够拿到两个结果了，而且可以确保二者之间不会有相互的影响。但是这需要选择恰当的成像方式。除了ECL为底物+预带HRP酶的二抗这个成像系统外，还有预载了荧光蛋白的二抗（同免疫荧光所用的抗体道理一样），配合检测不同波长的成像系统，最后实现在同一张膜上带有两种甚至三种信号的可能，进一步提升实验效率。

好，我就在这里结束我今天的分享了。最后总结一下：因为分子生物学实验的轮转时间（turn-over）相对较长，所以作为不断学习进步的研究学者，我们除了要不断学习、尝试、完善新技术以外，更重要是不断思考如何简化，甚至提升实验工作的效率。

最后再一次感谢BioEngX提供这个平台。祝大家实验顺利。

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作者: Viktor. Z

Viktor，于2013年本科毕业于纽卡斯尔大学，主攻生物化学与免疫学，随后加入了纽卡斯尔大学下属的细胞与分子研究院－核糖体生物学研究组（Ribosome Biology Group）开始了博士学业



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