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Western Blot 常见问题和处理方法

发布: 2016-02-21 1,202阅读 155评论

Western blot 结果中背景较高 (high background):

  • 膜封闭不够/封闭液 （blocking buffer）不适合：延长封闭时间，增加封闭液的浓度；尝试不同种类的封闭液，对比结果后选出效果最佳的。

  • 洗膜不够彻底：增长洗涤时间；增加洗涤剂的浓度。

  • 一抗/二抗 (primary/secondary antibodies) 稀释度不适宜：对抗体进行滴度测试 （titre test），选择最适宜的抗体稀释度。

  • 一抗是多克隆抗体 (polyclonal antibody): 特异性降低，可能与其他蛋白结合，建议换用单克隆抗体 （monoclonal antibody）。

  • 膜在实验过程中干过: 实验过程中要切记保持膜的湿润。

  • 检测时曝光时间过长: 减少曝光时间。

结果中杂带较多 (multiple/non-specific bands):

  • 目的蛋白有多个修饰位点（磷酸化位点 （phosphorylation site）、糖基化位点 (glycosylation site)、乙酰化位点 (acetylation site) 等），本身可以呈现多条带。

  • 样本处理过程中目的蛋白发生降解 (protein degradation): 加入蛋白酶抑制剂 (protease inhibitor)；样本处理时在冰上操作; 避免/减少样本的冻融循环 （free-and-thaw cycle）。

  • 样本处理过程中目的蛋白发生聚集（protein aggregation）：增加DTT的浓度；增长加热的时间。

  • 上样量（sample loading）/ 样本浓度过高：适当减少上样量/样本浓度。

  • 一抗是多克隆抗体 (polyclonal antibody): 特异性降低，可能与其他蛋白结合，建议换用单克隆抗体 （monoclonal antibody）。

  • 一抗/二抗不纯：纯化抗体；购买高纯度抗体。

  • 一抗或者二抗浓度偏高：降低抗体浓度，对抗体进行滴度测试 （titre test），选择最适宜的抗体稀释度。

  • 膜封闭不够/封闭液 （blocking buffer）不适合：延长封闭时间，增加封闭液的浓度；尝试不同种类的封闭液，对比结果后选出效果最佳的。

  • 洗膜不够彻底：增长洗涤时间；增加洗涤剂的浓度。

结果中无信号或显示信号弱:

  • 检测样本没有表达目的蛋白：选择表达量高的细胞作为阳性对照，用于确定检测样本是否为阴性。

  • 检测样本低表达目的蛋白：提高上样量/样本浓度，裂解液 （lysis buffer）中记得加入蛋白酶抑制剂。

  • 转移不完全或过转移：可以用丽春红（Ponceau S）染膜并结合考马斯亮蓝（Coomassie brilliant blue）染胶来确定条带是否成功转渍到膜上或是转移过度。根据结果可以适当调整转膜的时间和电流。

  • 抗体不能识别测试种属的相关蛋白：购买抗体前要认真阅读抗体说明书，确定是否能够交叉识别测试种属的对应蛋白。

  • 一抗孵育时间不足：建议放在4℃过夜结合。

  • 二抗与一抗不匹配：检查并且选择正确的针对一抗来源的种属的抗体。

  • 洗膜过度：洗膜时间不宜过长，加入的去垢剂 （detergent）不宜过强或过多，建议使用0.05-0.1%的弱去垢剂Tween-20。

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