Department of Neurobiology, Physiology, and Behavior, University of California, Davis, CA, USA.
Department of Neurobiology, Physiology, and Behavior, University of California, Davis, CA, USA; Department of Physiology and Membrane Biology, University of California, Davis, CA, USA.
Anal Biochem. 2019 Jun 15;575:44-53. doi: 10.1016/j.ab.2019.03.010. Epub 2019 Mar 23.
Normalization of Western blotting data is a critical step that is needed to reduce errors caused by unequal sample loading across lanes in a gel, inconsistent sample preparation, and variations due to experimental errors. Several papers have suggested that total protein normalization may be better than housekeeping protein normalization for Western blotting normalization. Ponceau S is the most commonly used stain for total protein normalization. A review of the literature and commercial websites suggest a multitude of Ponceau S staining protocols for total protein staining of blots. In this study, we explored which Ponceau S staining protocol would result in the highest sensitivity of protein band detection. Unexpectedly, we found that irrespective of the Ponceau S concentration (between 0.001 and 2% (w/v)), acid concentration, and acid type (acetic acid, trichloroacetic acid and/or sulfosalicylic acid), the sensitivity of protein detection remained constant. The most commonly used concentration of Ponceau S is 0.1%, while 0.001% (100-fold less) Ponceau S resulted in the same sensitivity of protein band detection. We suggest the use of the relatively inexpensive 0.01% Ponceau S in 1% acetic acid stain for total protein normalization as it is as effective as all the expensive formulations that are currently used.
Western blot 数据的归一化是一个关键步骤，它可以减少由于凝胶条带中样品加载不均、样品制备不一致以及实验误差引起的误差。有几篇论文表明，总蛋白归一化可能优于管家蛋白归一化用于 Western blot 归一化。丽春红 S 是最常用于总蛋白归一化的染色剂。文献综述和商业网站表明，有多种丽春红 S 染色方案可用于印迹的总蛋白染色。在这项研究中，我们探讨了哪种丽春红 S 染色方案会导致蛋白条带检测的灵敏度最高。出乎意料的是，我们发现无论丽春红 S 的浓度（0.001% 到 2%（w/v））、酸的浓度和酸的类型（乙酸、三氯乙酸和/或磺基水杨酸）如何，蛋白检测的灵敏度都保持不变。最常用的丽春红 S 浓度为 0.1%，而 0.001%（低 100 倍）的丽春红 S 也能达到相同的蛋白条带检测灵敏度。我们建议使用相对便宜的 0.01%丽春红 S 在 1%乙酸染色中进行总蛋白归一化，因为它与目前使用的所有昂贵配方一样有效。
