关于蛋白质免疫印迹（Western Blot，即 WB），想必做蛋白实验的小伙伴们都不会陌生，作为免疫学中最常用的一种实验方法，其基本原理是通过特异性抗体对凝胶电泳处理过的细胞或生物组织样品进行着色。通过分析条带大小和着色深度获得特定蛋白质在所分析的细胞或组织中表达情况的信息。
但是或许原理大家都懂，实验做起来却并不容易，每次当我们准备好样本，满心以为自己可以做出这样的实验结果时?：
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那么为了避免出现以上问题，下面就由小编给大家梳理一下 WB 中常见的问题以及解决方法。
一、信号强度低
信号强度低是指在正常曝光条件下得到的目的条带微弱或是没有目的条带，而增加曝光时间又会导致背景高、杂带多等问题。排除试剂质量、操作失误等方面的影响，这一类问题产生的主要原因如下：
① 样本蛋白表达量不足。建议添加蛋白表达量较高的细胞系作为阳性对照，或是适当增加上样量以获得较高水平的信号；
② 蛋白质降解。在蛋白样品制备期间注意蛋白酶抑制剂 PMSF 的使用，所制备的样品尽快检测或妥善保存；
③ 蛋白的转膜。使用适当孔径的膜，同时优化转膜时间，对于高分子量蛋白可能需要更长的转膜时间。
④ 抗体的使用。抗体的反应种属与实验类型需要能够满足实验的需求，此外，抗体的稀释比、孵育时间等问题也需要在试验中不断优化；
⑤ 蛋白与抗体结合率低。减少洗膜次数或缩短洗膜时间，降低洗膜液中 NaCl 浓度等；
⑥ 抗原被封闭液遮蔽。换用不同的封闭液，优化封闭液中蛋白质浓度并缩短封闭时间；
⑦ 甲醇浓度过高。会导致蛋白质与 SDS 分离，并沉淀在凝胶中，同时使凝胶收缩或变硬，从而抑制高分子量蛋白的转移。实验中可适当降低甲醇浓度或者使用乙醇、异丙醇代替。
二、非特异性条带或条带位置不对
WB 结果中目的条带之外的条带被称为非特异性条带，有时非特异性条带很明显，却没有目的条带，这些情况对结果分析会产生很大的干扰。一般来说引起这类问题的主要因素有：
① 抗体的非特异性结合。通常认为，多抗的特异性不如单抗，更容易产生非特异性条带，而适当降低抗体的浓度有助于减少杂带。同时为了排除二抗非特异性结合的可能，建议设二抗阴性对照；
② 样品蛋白量过高。适当降低上样量，同时延长洗涤时间与封闭时间可避免蛋白量过高对实验结果的不良影响；
③ 蛋白质降解。会导致条带位置变化并产生更多杂带，建议采用新鲜制备的或是冻融次数较少的样品；
④ 细胞传代次数过多。会导致蛋白表达模式的分化，建议使用原始或传代少的细胞株；
⑤ 蛋白存在复合物。有的目的蛋白会和其他蛋白结合形成复合物，导致条带位置改变，需要查阅相关文献来确定蛋白是否会形成稳定复合物；
⑥ 蛋白存在剪接体或多种修饰形式。部分蛋白存在剪切位点，有的剪切体具有免疫活性，在 WB 中也会被检测出。此外有的目的蛋白会存在糖基化位点或其他修饰位点，条带大小可能会远超理论分子量。因此需要查阅文献或是 uniprot 来得知蛋白的剪切体大小以及修饰位点等。
三、背景过高
在部分 WB 结果中，条带之外本应是空白的背景也会有较深的颜色，对分析结果会产生干扰，而且结果图不美观，难以用于文章发表。这种问题的可能原因有以下几点：
① 封闭不充分。背景过高的主要原因之一是封闭有问题，建议使用合适的封闭剂（脱脂奶粉、BSA 等），优化反应浓度与封闭时间，同时注意避免出现抗体与封闭剂的交叉反应，以及封闭剂与含有目标抗原，内源性生物素或者与抗生物素蛋白/链霉亲和素不相溶等问题；
② 洗膜不充分。适当增加洗膜次数和缓冲液体积，提高吐温20的百分比可改善由洗膜不充分导致的背景高的问题；
③ 抗体的使用。一抗浓度过高是高背景的原因之一，且二抗也存在与封闭剂非特异性结合的可能，因此建议适当优化一抗浓度，并设二抗阴性对照；
④ 曝光时间长。建议在实验中采用合适的曝光时间。
四、条带弥散或形状怪异
有时 WB 结果图中条带位置符合预期，但却因为条带弥散、形状怪异等因素导致无法使用，出现这种情况多半是制胶或电泳过程出了问题，具体如下：
① 电泳时电压、电流过高。会导致条带迁移速率过快以及 SDS-PAGE 温度升高，并可能使得条带弥散、形状变形。建议使用合适的电泳条件并保持低温；
② 电极不平衡。会导致条带偏斜，上样时在无样品的胶孔中加入等量样品缓冲液可避免这种情况；
③ 上样量过高，样品中盐离子浓度较大。上样量过高可能会导致条带弥散，样品中的盐离子浓度不一致则会导致条带不齐等问题。因此在上样时需保证样品量一致且适当，并保持相同的、较低的盐离子浓度。
④ 制胶问题。在配胶时一定要保证充分混匀，均匀凝胶，上样前用针头将胶孔拨正并吹出胶孔中的气泡。
⑤ 电泳缓冲液存放过久。电泳实验中的缓冲液如果放置过久也会对结果有不良影响，因此建议及时更换新的缓冲液。
五、膜上散布不均匀的污点
有时在 WB 做出结果后，除了目的条带以外，膜上还分布着不规律的污点，对结果也会有较大的干扰。这类问题产生的主要原因有以下几点：
① 试剂、仪器等污染。建议在每次实验前后注意清理实验仪器，同时及时更换出现问题的试剂；
② 转膜时有气泡。确保在转膜过程中将气泡排除干净；
③ 抗体孵育时与膜接触不充分。确保在抗体孵育过程中，液体总量足够，同时将抗体均匀配置，并在摇晃的条件下进行孵育；
④ 曝光时间。过度曝光会导致斑点更明显，建议采用合适的曝光时间；
⑤ 膜干燥。实验中要保证有充分的反应液，避免出现干膜现象。
以上就是本次分享的全部内容，看完这一大堆之后是不是觉得头都大了？那么有没有什么方法能够简单、省心还能得到好的实验结果呢？
当然有！为了帮助大家做出更美观 WB 图，诺禾致源最新推出了的 simple Western 服务，像这样的 WB 结果图想不想要？
简洁、美观同时便于数据分析，目前已获得包括《Nature》、《Science》、《Cell》在内的多个顶级期刊的认可。
了解更多，发送邮件至 novoab@novogene.com 进行咨询。
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