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Western blot离完美总差一步，可能因这7个小误区

发布: 2018-08-13 731阅读 195评论

遥想当年，小编初接触WB时，只顾一味瞎折腾，完美地避开了所有正确操作，以至于每次做完该实验，就觉得自己的血槽已经被耗光了。好在多次与WB斗智斗勇后，也积攒了不少经验，这就将wb中常见的7大错误分享给大家，与君共勉。

1. 乌糟糟的蛋白样品

“良好的开始是成功的一半”，其实，对于WB这座大厦而言，好的蛋白样品的准备无疑是打下了良好的地基，而坏的样品则奠定了“豆腐渣”工程的基调。因而，正确的蛋白质提取和样品制备是至关重要的。

尽管每个实验室都有相应的样品准备方法，但做实验的小伙伴在制备蛋白样品时仍需要考虑以下因素：盐离子浓度、缓冲液种类、抑制剂、还原剂和加热时间等。一个最佳Protocol应该最大程度上提高蛋白质的溶解度和稳定性，有助于WB更好得可视化目的蛋白。

2. 失败的凝胶制备

上样前，要谨记完美的凝胶和好的样品才是最配的，不然只能使之前的种种努力付之东流。为了避免前功尽弃，在使用SDS-PAGE凝胶前需要仔细检查并警惕以下情况出现：

1）凝胶中不仅出现气泡，而且孔道之间并不均一，这会严重影响蛋白质在凝胶中的迁移。

2）当凝胶边缘开始收缩时，说明凝胶已经变得非常干燥，需要重新配制。

3）凝胶凝固时间太短，仍处于可以流动状态。为了确保凝胶完全凝固，可将胶轻轻倾斜来进行检查。

3. 转膜中的层层“陷阱”

WB中，转膜是对Western最终结果影响最大的一步，转膜质量的好坏直接决定了实验结果的好坏。那么转膜中又存在哪些容易被人忽视的陷阱存在呢？

1）徒手抓蛋白转移膜。毫无疑问，此时手指上的油脂与蛋白会封闭转移膜，容易产生背景污斑影响实验结果。因此建议实验全程要用手套及镊子。

2）忽略了分离胶的上下方向以及膜的正反面。通常使用预染Marker可解决此问题，此外还可以将胶和膜的一角剪掉来进行区分。

3）转移膜和凝胶不幸干涸了。一般转膜前可将凝胶放在缓冲液里平衡一下防止胶变形，也有助于进一步去掉有碍转膜的杂质。也可在这一步通过浸泡对蛋白复性并直接检测蛋白活性。

4）转移缓冲液中甲醇浓度过高，可使蛋白质与SDS分离，从而沉淀在凝胶中，同时也会使凝胶收缩或变硬，抑制高分子量蛋白的转移。此时可降低甲醇浓度或用乙醇或异丙醇代替。

4. 转膜后检测结果一团糟

通常为了检测转膜是否成功，可用丽春红进行染色。即将膜放入TBST中清洗一次，而后将其置于丽春红染色工作液中，室温下摇动染色5min并用大量水洗膜，直至水变清无色，蛋白条带清晰。

同样，一些让人头疼的问题也随之而来。丽春红染色后，目的蛋白条带常常黯淡无光，甚至有时不翼而飞。而这些大多是由以下原因造成的：

1）蛋白转膜的时间太短或者处于热度较高的环境之中，此时只需在一个较低的电压下转膜较长的时间即可；

2）在处理“滤纸-凝胶-膜-滤纸”的三明治时，使得膜和胶与对方的背后滤纸相接触导致电流不能通过膜，从而转膜无效。因而，三明治结构中，膜、胶、滤纸的大小最好是一样的。

5. 不合适的抗体（一抗&二抗）

对于国内的大多数实验室来讲，做western blot实验选择抗体是个头疼的问题。原因很简单，买进口抗体捉襟见肘，买国产抗体得需要大无畏的勇气。然而要想与WB实验进行愉快的玩耍，就一定要确保抗体的高质量，此时一些可信赖的品牌如Proteintec Group、Epitomics、Abgent、Abnova等便是不错的选择。

另外，还有个最安全可靠的办法就是直接参考文献中使用的抗体，但是仍需要注意3点：一是文章的可靠性，要参考IF高于3分的文章；二是实验必须是同一类实验，别人做IHC的抗体做WB是没有意义的；三是检测的是内源蛋白还是外源蛋白，一般只有内源蛋白的Western blot才有参考意义，但如果同时做了过表达和敲减，也是可以的。

6. 抗体因保存不当而降解

抗体无论是保存还是运输都应尽可能的避免反复冻融。收到抗体后，无论是液体还是冻干粉状态，请务必在4℃,12000rpm,离心3分钟，，再打开管盖进行分装和保存（如果抗体体积小于50ul，请延长离心时间至5分钟，以保证全部抗体均离心下来）。

抗体分装的量以一次实验用完为好，最少不能少于10ul每份。因为分装体积越小，抗体的浓度越可能会受到蒸发及管壁吸附的影响。复融后的分装抗体如果一次用不完，将剩余母液保存在4℃，避免再次冻起来。抗体工作液应该现配现用，4℃保存尽量不要超过1天。

抗体中的叠氮化钠（终浓度0.02% (w/v)，65Da）尽管会防止微生物污染，但仍会对实验产生影响。又因IgG的分子量是150kDa（IgM约600kDa），可使用截留分子量为14kDa的滤膜将叠氮化钠从抗体中去除。

7. 发光实验中背景太高

一般，在很短时间里曝光产生高背景多是由含HRP-抗体浓度过高，洗涤不充分或封闭不充分造成的。另外，如果发光试剂不好，也会制造背景颜色较深，这种情况在较长时间曝光的情况下更为明显。

因而，WB曝光时要注意掌控好时间，时间太长会使得背景深，过短又会不容易曝出条带或曝出条带很浅，所以需要优化出适合自己靶蛋白和实验体系的最佳曝光时间。

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