Epicocconone staining: A powerful loading control for Western blots
Epicocconone staining: A powerful loading control for Western blots
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DOI：
10.1002/pmic.201300089
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发表时间：
2014-02-01
期刊：
影响因子：
3.4
通讯作者：
Friauf, Eckhard
中科院分区：
文献类型：
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作者：
Moritz, Christian P.;Marz, Sabrina X.;Friauf, Eckhard
蛋白质印迹分析通常被用来量化样品之间蛋白质水平的差异。为了控制等负荷和算术补偿负荷差异，通常使用管家蛋白的免疫检测。由于潜在的偏见，这种方法受到了批评。在这里，我们评估基于表康酮的总蛋白染色(E-TOPS)作为一种替代方法。我们将其与另外两种总蛋白染色(Coomassie和SyproRuby)以及管家蛋白(微管蛋白和甘油醛3-磷酸脱氢酶)的免疫检测进行了比较。评价包括天然和合成的表康酮化合物。这两种化合物的结果高度一致，并且显示出比其他变体更敏感(1g)和更少变化的染色特性。E-TOPS的高灵敏度，覆盖了微量的蛋白质，使其成为一种强大的加载控制，特别是对于珍贵的样品。在生物学和技术差异方面，E-TOPS优于微管蛋白和甘油醛3-磷酸脱氢酶免疫染色。此外，E-TOPS对随后的免疫检测没有影响，允许在免疫检测之前及早控制适当的负荷。与以前的研究相比，我们发现了E-TOPS的对数染色特性，当使用它进行算术归一化时，应该考虑这一点。总而言之，我们展示了E-TOPS作为Western blots的加载控制的优越能力。
Western blot analysis is routinely employed for quantifying differences in protein levels between samples. To control equal loading and to arithmetically compensate loading differences, immunodetection of housekeeping proteins is commonly used. Due to potential biases, this approach has been criticized. Here, we evaluate epicocconone-based total protein staining (E-ToPS) as an alternative. We compared it with two other total protein stainings (Coomassie and Sypro Ruby) and with immunodetection of housekeeping proteins (-tubulin and glyceraldehyde 3-phosphate dehydrogenase). Evaluation comprised both the natural and the synthetic epicocconone compound. Both compounds produced highly congruent results and showed more sensitive ( 1 g) and less variable staining properties than the other variants. The high sensitivity of E-ToPS, covering minute protein amounts, makes it a powerful loading control, especially for precious samples. Regarding biological and technical variances, E-ToPS outperformed immunostaining against -tubulin and glyceraldehyde 3-phosphate dehydrogenase. Furthermore, E-ToPS had no impact on subsequent immunodetection, allowing for an early control of proper loading prior to immunodetection. In contrast to earlier studies, we found logarithmic staining properties for E-ToPS, which should be considered when using it for arithmetic normalization. In conclusion, we demonstrate the superior power of E-ToPS as a loading control for Western blots.